Peptide compositions

ABSTRACT

Described are treatments of disease wherein a gene-regulatory peptide as provided herein is useful as a modulator of NF-kappaB. Also described is a pharmaceutical composition for topical application comprising a gene-regulatory peptide or functional analogue thereof, wherein the peptide or analogue modulates translocation and/or activity of a gene transcription factor.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of the U.S. patent application Ser. No. 10/409,659, filed Apr. 8, 2003, pending, which is a continuation-in-part of U.S. patent application Ser. No. 10/028,075, filed Dec. 21, 2001, now U.S. Pat. No. ______, the contents of the entirety of each of which are incorporated herein by this reference.

TECHNICAL FIELD

The current invention relates generally to biotechnology, and, more particularly, to the body's innate way of modulating important physiological processes. It builds on insights reported in WO 99/59717, WO 01/00259 and PCT/NL/00639 receive and review correspondence from Dr. Wensvoort; prepare continuation-in-part patent application. U.S. Pat. No. 5,380,668 to Herron (Jan. 10, 1995), the contents of the entirety of which are incorporated by this reference, discloses, among other things, various compounds having the antigenic binding activity of hCG. Herron further discloses means and methods for making oligopeptides.

BACKGROUND

In these earlier patent applications, small gene-regulatory peptides are described that are present naturally in pregnant women and are derived from proteolytic breakdown of placental gonadotropins such as human chorionic gonadotropin (hCG) produced during pregnancy. These peptides (in their active state often only at about four to six amino acids long) were shown to have unsurpassed immunological activity that they exert by regulating expression of genes encoding immunomodulatory and inflammatory mediators such as cytokines. Surprisingly, it was found that breakdown of hCG provides a cascade of peptides that help maintain a pregnant woman's immunological homeostasis. These peptides are nature's own substances that balance the immune system to assure that the mother stays immunologically sound while her fetus does not get prematurely rejected during pregnancy but instead is safely carried through its time of birth.

Where it was generally thought that the smallest breakdown products of proteins have no specific biological function on their own (except to serve as antigen for the immune system), from the above three patent applications, it now emerges that the body in fact routinely utilizes the normal process of proteolytic breakdown of the proteins it produces to generate important gene-regulatory compounds, short peptides that control the expression of the body's own genes. Apparently the body uses a gene-control system ruled by small broken down products of the exact proteins that are encoded by its own genes.

During pregnancy, the maternal system introduces a status of temporary immuno-modulation which results in suppression of maternal rejection responses directed against the fetus. Paradoxically, during pregnancy, often the mother's resistance to infection is increased and she is found to be better protected against the clinical symptoms of various auto-immune diseases such as rheumatism and multiple sclerosis. The protection of the fetus can thus not be interpreted only as a result of immune suppression. Each of the above three applications have provided insights by which the immunological balance between protection of the mother and protection of the fetus can be understood.

Inventors hereof have shown that certain short breakdown products of hCG (i.e., short peptides which can easily be synthesized, if needed modified, and used as pharmaceutical composition) exert a major regulatory activity on pro- or anti-inflammatory cytokine cascades that are governed by a family of crucial transcription factors, the NFkappaB family which stands central in regulating the expression of genes that shape the body's immune response.

Most of the hCG produced during pregnancy is produced by cells of the placenta, the exact organ where cells and tissues of mother and child most intensely meet and where immuno-modulation is most needed to fight off rejection. Being produced locally, the gene-regulatory peptides which are broken down from hCG in the placenta immediately balance the pro- or anti-inflammatory cytokine cascades found in the no-mans land between mother and child. Being produced by the typical placental cell, the trophoblast, the peptides traverse extracellular space; enter cells of the immune system and exert their immuno-modulatory activity by modulating NFkappaB-mediated expression of cytokine genes, thereby keeping the immunological responses in the placenta at bay.

SUMMARY OF THE INVENTION

It is herein postulated that the beneficial effects seen on the occurrence and severity of auto-immune disease in the pregnant woman result from an overspill of the hCG-derived peptides into the body as a whole; however, these effects must not be overestimated, as it is easily understood that the further away from the placenta, the less immuno-modulatory activity aimed at preventing rejection of the fetus will be seen, if only because of a dilution of the placenta-produced peptides throughout the body as a whole. However, the immuno-modulatory and gene-regulatory activity of the peptides should by no means only be thought to occur during pregnancy and in the placenta; man and women alike produce hCG, for example in their pituitaries, and nature certainly utilizes the gene-regulatory activities of peptides in a larger whole.

Consequently, a novel therapeutic inroad is provided, using the pharmaceutical potential of gene-regulatory peptides and derivatives thereof. Indeed, evidence of specific up- or down-regulation of NFkappaB driven pro- or anti-inflammatory cytokine cascades that are each, and in concert, directing the body's immune response was found in silico in gene-arrays by expression profiling studies, in vitro after treatment of immune cells and in vivo in experimental animals treated with gene-regulatory peptides. Also, considering that NF-kappaB is a primary effector of disease (A. S. Baldwin, J. Clin. Invest., 2001, 107:3-6), using the hCG-derived gene-regulatory peptides offer significant potential for the treatment of a variety of human and animal diseases, thereby tapping the pharmaceutical potential of the exact substances that help balance the mother's immune system such that her pregnancy is safely maintained. The compounds according to the general formula may be prepared in a manner conventional for such compounds. To that end, suitably N alpha protected (and side-chain protected if reactive side-chains are present) amino acid derivatives or peptides are activated and coupled to suitably carboxyl protected amino acid or peptide derivatives either in solution or on a solid support.

Protection of the alpha-amino functions generally takes place by urethane functions such as the acid-labile tertiary-butyloxycarbonyl group (“Boc”), benzyloxycarbonyl (“Z”) group and substituted analogs or the base-labile 9-fluoremyl-methyloxycarbonyl (“Fmoc”) group. The Z group can also be removed by catalytic hydrogenation. Other suitable protecting groups include the Nps, Bmv, Bpoc, Aloc, MSC, etc. A good overview of amino protecting groups is given in The peptides, Analysis, Synthesis, Biology, Vol. 3, E. Gross and J. Meienhofer, eds. (Academic Press, New York, 1981). Protection of carboxyl groups can take place by ester formation, for example, base-labile esters like methyl or ethyl, acid labile esters like tert. butyl or, substituted, benzyl esters or hydrogenolytically. Protection of side-chain functions like those of lysine and glutamic or aspartic acid can take place using the aforementioned groups. Protection of thiol, and although not always required, of guanidino, alcohol and imidazole groups can take place using a variety of reagents such as those described in The Peptides, Analysis, Synthesis, Biology, id. or in Pure and Applied Chemistry, 59(3), 331-344 (1987). Activation of the carboxyl group of the suitably protected amino acids or peptides can take place by the azide, mixed anhydride, active ester, or carbodiimide method especially with the addition of catalytic and racemization-suppressing compounds like 1-N-N-hydroxybenzotriazole, N-hydroxysuccinimide, 3-hydroxy-4-oxo-3,4-dihydro-1,2,3,-benzotria-zine, N-hydroxy-5 norbornene-2,3-dicar-boxyimide. Also the anhydrides of phosphorus based acids can be used. See, e.g., The Peptides, Analysis, Synthesis, Biology, supra, and Pure and Applied Chemistry, 59(3), 331-344 (1987).

It is also possible to prepare the compounds by the solid phase method of Merrifield. Different solid supports and different strategies are known, see, e.g., Barany and Merrifield in The Peptides, Analysis, Synthesis, Biology, Vol. 2, E. Gross and J. Meienhofer, eds. (Acad. Press, New York, 1980), Kneib-Cordonier and Mullen Int. J. Peptide Protein Res., 30, 705-739 (1987); and Fields and Noble Int. J. Peptide Protein Res., 35, 161-214 (1990). The synthesis of compounds in which a peptide bond is replaced by an isostere, can, in general, be performed using the previously described protecting groups and activation procedures. Procedures to synthesize the modified isosteres are described in the literature, for instance, for the —CH₂—NH— isostere and for the —CO—CH₂— isostere.

Removal of the protecting groups, and, in the case of solid phase peptide synthesis, the cleavage from the solid support, can take place in different ways, depending on the nature of those protecting groups and the type of linker to the solid support. Usually deprotection takes place under acidic conditions and in the presence of scavengers. See, e.g., Volumes 3, 5, and 9 of the series on The Peptides Analysis, Synthesis, Biology, supra.

Another possibility is the application of enzymes in synthesis of such compounds; for reviews see, e.g., H. D. Jakubke in The Peptides, Analysis, Synthesis, Biology, Vol. 9, S. Udenfriend and J. Meienhofer, eds. (Acad. Press, New York, 1987).

Although possibly not desirable from an economic point of view, oligopeptides hereof could also be made according to recombinant DNA methods. Such methods involve the preparation of the desired oligopeptide thereof by means of expressing recombinant polynucleotide sequence that codes for one or more of the oligopeptides in question in a suitable microorganism as host. Generally the process involves introducing into a cloning vehicle (e.g., a plasmid, phage DNA, or other DNA sequence able to replicate in a host cell) a DNA sequence coding for the particular oligopeptide or oligopeptides, introducing the cloning vehicle into a suitable eukaryotic or prokaryotic host cell, and culturing the host cell thus transformed. When a eukaryotic host cell is used, the compound may include a glycoprotein portion.

As used herein, a “functional analogue” or “derivative” of a peptide includes an amino acid sequence, or other sequence monomers, which has been altered such that the functional properties of the sequence are essentially the same in kind, not necessarily in amount. An analogue or derivative can be provided in many ways, for instance, through “conservative amino acid substitution.” Also peptidomimetic compounds can be designed that functionally or structurally resemble the original peptide taken as the starting point but that are for example composed of non-naturally occurring amino acids or polyamides. With “conservative amino acid substitution,” one amino acid residue is substituted with another residue with generally similar properties (size, hydrophobicity), such that the overall functioning is likely not to be seriously affected. However, it is often much more desirable to improve a specific function.

A derivative can also be provided by systematically improving at least one desired property of an amino acid sequence. This can, for instance, be done by an Ala-scan and/or replacement net mapping method. With these methods, many different peptides are generated, based on an original amino acid sequence but each containing a substitution of at least one amino acid residue. The amino acid residue may either be replaced by alanine (Ala-scan) or by any other amino acid residue (replacement net mapping). This way, many positional variants of the original amino acid sequence are synthesized. Every positional variant is screened for a specific activity. The generated data are used to design improved peptide derivatives of a certain amino acid sequence.

A derivative or analogue can also be, for instance, generated by substitution of an L-amino acid residue with a D-amino acid residue. This substitution, leading to a peptide that does not naturally occur in nature, can improve a property of an amino acid sequence. It is, for example, useful to provide a peptide sequence of known activity of all D-amino acids in retro inversion format, thereby allowing for retained activity and increased half-life values. By generating many positional variants of an original amino acid sequence and screening for a specific activity, improved peptide derivatives comprising such D-amino acids can be designed with further improved characteristics.

A person skilled in the art is well able to generate analogous compounds of an amino acid sequence. This can, for instance, be done through screening of a peptide library. Such an analogue has essentially the same functional properties of the sequence in kind, not necessarily in amount. Also, peptides or analogues can be circularized, for example, by providing them with (terminal) cysteines, dimerized or multimerized, for example, by linkage to lysine or cysteine or other compounds with side-chains that allow linkage or multimerization, brought in tandem- or repeat-configuration, conjugated or otherwise linked to carriers known in the art, if only by a labile link that allows dissociation.

As used herein, an oligopeptide also includes, for example, an acceptable salt, base, or ester of the oligopeptide or a labeled oligopeptide. As used herein, “acceptable salt” refers to salts that retain the desired activity of the oligopeptide or equivalent compound, but preferably do not detrimentally affect the activity of the oligopeptide or other component of a system in which uses the oligopeptide. Examples of such salts are acid addition salts formed with inorganic acids, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, and the like. Salts may also be formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, and the like. Salts may be formed with polyvalent metal cations such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel and the like or with an organic cation formed from N,N′-dibenzylethylenediamine or ethylenediamine, or combinations thereof (e.g., a zinc tannate salt).

The oligopeptide, or its modification or derivative, can be administered as the entity, as such, or as a pharmaceutically acceptable acid- or base addition salt, formed by reaction with an inorganic acid (such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid); or with an organic acid (such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid); or by reaction with an inorganic base (such as sodium hydroxide, ammonium hydroxide, potassium hydroxide); or with an organic base (such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines). A selected peptide and any of the derived entities may also be conjugated to sugars, lipids, other polypeptides, nucleic acids and PNA; and function in-situ as a conjugate or be released locally after reaching a targeted tissue or organ.

A pharmaceutical composition for use herein may be administered to the subject parenterally or orally. Such a pharmaceutical composition may consist essentially of (or consist of) oligopeptide and PBS. It certain embodiments, the oligopeptide is of synthetic origin. Suitable treatment, for example, entails administering the oligopeptide (or salt or ester) in the pharmaceutical composition to the patient intravenously in an amount of from about 0.0001 to about 35 mg/kg body mass of the subject. It may be useful that the pharmaceutical composition consists essentially of from one to three different oligopeptides.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Hemorrhagic Shock model (HS) (*=time of administration peptide A, B or C in the peptide groups).

FIG. 2: Mean Arterial Pressure in sham, shock, and Peptide A, B and C experiments.

FIG. 3: Hematocrit in (from left to right) sham, shock, and Peptide A, B and C experiments.

FIG. 4: Leukocytes during sham, trauma-hemorrhage, pep A, B and C experiments.

FIG. 5: Macrophages (MO) and granulocytes (GR) in (from left to right) sham, trauma-hemorrhagic shock, and Peptide A, B and C experiments.

FIG. 6: Arterial blood flow in (from left to right) sham, shock, and Peptide A, B and C experiments.

FIG. 7: Hemorrhagic shock model. A) Schematic representation of the experimental design. B) The measured mmHg was recalculated in percentages to standardize the experiment and to compensate for animal differences. C) Percentage of leukocytes in blood during various time points of the experiment.

FIG. 8: TNF-α plasma levels in different experimental groups determined at 15 minutes before and 30, 60, 90, 120, 150 and 180 minutes after the onset of hemorrhagic shock. □ Sham, O HS, ∇ HS/LQGV, ⋄ HS/AQGV, Δ HS/LAGV. Each figure represents one animal.

FIG. 9: IL-6 plasma levels in different experimental groups determined at 120, 150 and 180 minutes after the onset of hemorrhagic shock □ Sham, O HS, ∇ HS/LQGV, ⋄ HS/AQGV, Δ HS/LAGV. Each figure represents one animal.

FIG. 10: Transcript levels for A) TNF-α, B) IL-6 and C) ICAM-1 in the liver, 180 minutes after the onset of hemorrhagic shock. Data expressed are correlated to GAPDH expression. □ Sham, O HS, ∇ HS/LQGV, ⋄ HS/AQGV, Δ HS/LAGV. Each figure represents one animal.

FIGS. 11A and 11B: Depict the same experiment but reflect the scores of two independent observers RK and JV. Peptide A=LAGV (SEQ ID NO:10); Peptide B=AQGV (SEQ ID NO:2); Peptide G=VLPALPQ (SEQ ID NO:13); Peptide I=LQGV (SEQ ID NO:1). Treatment protocol: daily application of 4% imiquimod cream (day 0-day 6) on shaved back; 300 μg/mouse peptide in PBS i.p. on days −1, 1, 3 and 5. Scoring for redness, scaling and skin thickness, daily, blindly. Cumulative score=redness+scaling+thickness (scale 0-12).

FIG. 12: Peptide I=LQGV (SEQ ID NO:1). Treatment protocol: daily application of 5% imiquimod cream (day 1-day 5) on shaved back and ear; immediately before imiquimod application, treat skin from back and ear with petroleum ether to remove fat and scales (also groups not treated with petroleum ether); 500 μg/mouse peptide I in PBS i.p. on day 1, 3 and 5; measuring ear thickness on days 1, 3, and 5. Scoring for redness, scaling and skin thickness, daily, blindly. Cumulative score=redness+scaling+thickness (scale 0-12)

DETAILED DESCRIPTION OF THE INVENTION

PCT/NL02/00639 also provides an insight into the biology and physiology of the nature of regulatory factors in gene regulation in cellular organisms that allows for the identification and development of an artificial or synthetic compound acting as a gene regulator, and its use as new chemical entity for the production of a pharmaceutical composition or its use in the treatment of disease. Many of small peptides are herein identified as short, gene regulatory peptides. These short, gene regulatory peptides are commonly from 2 to 15 amino acids long, but preferably shorter, e.g., from 3 to 12 amino acids, i.e., 4, 5, 6 or 7 amino acids long and are derivable by proteolytic breakdown of endogenous proteins of an organism, or are derivable by proteolytic breakdown of proteins of a pathogen, i.e., during the presence of the pathogen in a host organism, and act as a gene-regulatory peptide to cells of the organism, in that they can exert an often very specific gene regulatory activity on cells of the organism. In a particular embodiment, PCT/NL02/00639 provides specific gene-regulatory peptides and mechanisms allowing for therapeutically controlling NFKB-initiated gene expression, and thereby modulating pro- and anti-inflammatory cytokine expression under a variety of different conditions and circumstances.

Classically, many genes are regulated not by a gene-regulatory peptide that enters the cells, but by molecules that bind to specific receptors on the surface of cells. Interaction between cell-surface receptors and their ligands can be followed by a cascade of intracellular events including variations in the intracellular levels of so-called second messengers (diacylglycerol, Ca²⁺, cyclic nucleotides). The second messengers in turn lead to changes in protein phosphorylation through the action of cyclic AMP, cyclic GMP, calcium-activated protein kinases, or protein kinase C, which is activated by diaglycerol. Many of these classic responses to binding of ligands to cell-surface receptors are cytoplasmatic and do not involve immediate gene activation in the nucleus. Some receptor-ligand interactions, however, are known to cause prompt nuclear transcriptional activation of a specific and limited set of genes. However, progress has been slow in determining exactly how such activation is achieved. In a few cases, the transcriptional proteins that respond to cell-surface signals have been characterized.

One of the clearest examples of activation of a pre-existing inactive transcription factor following a cell-surface interaction is the nuclear factor (NF)-kappaB, which was originally detected because it stimulates the transcription of genes encoding immunoglobulins of the kappa class in B-lymphocytes. The binding site for NK-kappaB in the kappa gene is well defined (see, for example, P. A. Baeuerle and D. Baltimore, 1988, Science 242:540), providing an assay for the presence of the active factor. This factor exists in the cytoplasm of lymphocytes complexed with an inhibitor. Treatment of the isolated complex in vitro with mild denaturing conditions dissociates the complex, thus freeing NK-kappaB to bind to its DNA site. Release of active NF-kappaB in cells is now known to occur after a variety of stimuli including treating cells with bacterial lipopolysaccharide (LPS) and extracellular polypeptides as well as chemical molecules (e.g., phobol esters) that stimulate intracellular phosphokinases. Thus a phosphorylation event triggered by many possible stimuli may account for NF-kappaB conversion to the active state. The active factor is then translocated to the cell nucleus to stimulate transcription only of genes with a binding site for active NF-kappaB. In PCT/NL02/00639 it was shown that a variety of the short peptides as indicated above exert a modulatory activity on NF-kappaB activity.

The inflammatory response involves the sequential release of mediators and the recruitment of circulating leukocytes, which become activated at the inflammatory site and release further mediators (Nat. Med. 7:1294; 2001). This response is self-limiting and resolves through the release of endogenous anti-inflammatory mediators and the clearance of inflammatory cells. The persistent accumulation and activation of leukocytes is a hallmark of chronic inflammation. Current approaches to the treatment of inflammation rely on the inhibition of pro-inflammatory mediator production and of mechanisms that initiate the inflammatory response. However, the mechanisms by which the inflammatory response resolves might provide new targets in the treatment of chronic inflammation. Studies in different experimental models of resolving inflammation have identified several putative mechanisms and mediators of inflammatory resolution.

Considering that NF-kappaB is thought by many to be a primary effector of disease (A. S. Baldwin, J. Clin. Invest., 2001, 107:3-6), numerous efforts are herein provided to develop safe modulators of NF-kappaB to be used in treatment of both chronic and acute and systemic and local disease situations.

The administration of a gene-regulatory peptide may be done as a single dose, as a discontinuous sequence of various doses, or continuously for a period of time sufficient to permit substantial modulation of gene expression. In the case of a continuous administration, the duration of the administration may vary depending upon a number of factors which would readily be appreciated by those skilled in the art. A gene-regulatory peptide as provided herein is useful as a modulator of NF-kappaB to be used in treatment of both chronic and acute and systemic and local disease situations.

The administration dose of the gene-regulatory peptide may be varied over a fairly broad range. The concentrations of an active molecule which can be administered would be limited by efficacy at the lower end and the solubility of the compound at the upper end. The optimal dose or doses for a particular patient should and can be determined by taking into consideration relevant factors such as the condition, weight and age of the patient, and other considerations of the physician or medical specialist involved.

EXAMPLES

One example wherein a gene-regulatory peptide as provided herein is useful as a modulator of NF-kappaB to be used in treatment of disease is in topical applications. In one embodiment, the invention provides a pharmaceutical composition for topical application comprising a gene-regulatory peptide or functional analogue thereof, and use of a gene-regulatory peptide or functional analogue thereof for the production of a pharmaceutical composition for topical application. Such a composition is most useful to treat a particular surface area, such as a certain area of the skin or mucus membranes and thereby affects essentially only the area to which it is applied and some of the underlying tissue. Where the body logically responds to local insults by eliciting a (local) inflammation, the invention provides use of a gene-regulatory peptide comprising an NF-kappaB down-regulating peptide or functional analogue thereof for the production of a pharmaceutical composition for the topical treatment of a subject, to actually counter the inflammation and prevent systemic responses and overly active scar tissue formation.

The invention also provides a cream or ointment comprising an NF-kappaB down-regulating peptide or functional analogue thereof, and if so desired, a bactericidal or bacteriostatic compound or a compound comprising silver. Wound management will vary according to the depth of the insult. The true depth of the affected area will become more obvious with time and therefore the wound must be reassessed to ensure that wound management is appropriate. Systemic, and even topical, antibiotics are not to be used prophylactically, and are in general only appropriate when demonstrated infection is present, however, is in particular useful that translocation and/or activity of the NF-kappaB/Rel protein is inhibited to counter the local cytokine cascade leading to an inflammation by the inclusion of one or more of the NFkappaB down-regulating peptides or functional analogues thereof as identified herein, at a concentration of for example 1 to 1000 microg/g, preferably 50-300 microg/g, and at that time it is even more useful that the pharmaceutical composition for topical use is also provided with antibacterial compounds, such as compounds that comprise silver, such as a antibacterial cream or ointment comprising micronized silver sulfadiazine and an NFkappaB down-regulating peptide.

The invention thus provides a method to treat an injury of a subject wherein the subject is provided with a topical agent directed against a bacterial infection such as a bacteriostatic or bacteriocidal compound such as tetracycline or a sulfa compound wherein the topical agent also comprises a NFkappaB down-regulating peptide at a concentration of for example 1 to 1000 microg/g, preferably 50-300 microg/g. Typical other substances found in such a cream or ointment are 10 mg/gram of micronized silver sulfadiazine and a lege artis cream vehicle composed of white petrolatum, stearyl alcohol, isopropyl myristate, sorbitan monooleate, polyoxyl 40 stearate, propylene glycol, and water. Another anti-inflammatory and anti-infective cream for topical administration as herein provided comprises one or more of NFkappaB down-regulating peptides VLPALPQVVC (SEQ ID NO:_), LQGVLPALPQ (SEQ ID NO:_), LQG, LQGV (SEQ ID NO:1), AQGV (SEQ ID NO:2), LAGV (SEQ ID NO:10), VLPALPQ (SEQ ID NO:13), GVLPALPQ (SEQ ID NO:16), VLPALP (SEQ ID NO:4), VVC, MTR at a concentration of for example 50-300 microgram/gram and contains per gram mafenide acetate equivalent to 85 mg of the base. The cream vehicle for example consists of cetyl alcohol, stearyl alcohol, cetyl esters wax, polyoxyl 40 stearate, polyoxyl 8 stearate, glycerin, and water, with methylparaben, propylparaben, sodium metabisulfite, and edetate disodium as preservatives may be added.

Other useful pharmaceutical compositions for topical use are an oil-in-water emulsion base composed of glycerin, cetyl alcohol, stearic acid, glyceryl monostearate, mineral oil, polyoxyl 40 stearate, menthol, benzyl alcohol, and purified water, comprising a gene-regulatory peptide, preferably a NFkappaB down-regulating peptide at a concentration of, for example, 50-300 microgram/gram, a hydrophilic, emollient cream base of propylene glycol, white petrolatum, cetearyl alcohol, glyceryl stearate, PEG 100 stearate, monobasic sodium phosphate, chlorocresol, phosphoric acid, and purified water with gene-regulatory peptides at the above preferably concentrations, an ointment base of hexylene glycol, white wax, propylene glycol stearate, and white petrolatum with the peptides; an emollient cream base of purified water, chlorocresol; propylene glycol, white petrolatum, white wax, cyclomethicone; sorbitol solution, glyceryl oleate/propylene glycol, with gene-regulatory peptide; a lotion base of purified water, isopropyl alcohol, hydroxypropyl cellulose, propylene glycol, sodium phosphate monobasic monohydrate, phosphoric acid, preferably used to adjust the pH to 4.5, and gene regulatory peptide; polyethylene glycol ointment, and gene-regulatory peptide; a topical gel, containing the active ingredient gene-regulatory peptide at 25-250 mg/g and the following inactive ingredients: sodium chloride, sodium acetate trihydrate, glacial acetic acid, water for injection.

In another example wherein a gene-regulatory peptide as provided herein is useful as a modulator of NF-kappaB to be used in treatment of disease the invention provides a pharmaceutical composition for topical application to the eye or ear comprising a gene-regulatory peptide or functional analogue thereof, and use of a gene-regulatory peptide or functional analogue thereof for the production of a pharmaceutical composition for application to the eye or ear. Such a composition is most useful to treat a surface area, such as the conjunctivae of the eyes, or the lining of the tympanic cavity.

In a further embodiment, provided is a pharmaceutical composition comprising a gene regulatory peptide or functional equivalent thereof for the treatment of a conjunctivitis, notably for the treatment of a so called dry eye. Dry eye is the result of any number of unrelated causes or conditions. Some of the most common factors that contribute to dry eye include: Dry or dusty environment, aging, hormonal changes, autoimmune diseases such as seen with Sjogren's disease, certain types of medications and contact lens wear. Regardless of the cause, all dry eye patients have in common abnormal or insufficient tears. This leads to reduced tear clearance, increased osmolarity, ocular surface irritation, and the infiltration and production of pro-inflammatory cytokines. The end result is inflammation. Once inflammation starts, damage can occur to ocular structures that perpetuate and intensify a cycle of signs, symptoms and further inflammation. To counter this inflammation, the invention provides a pharmaceutical composition comprising a gene-regulatory peptide for the treatment of dry eye or other conjunctivitis. Such an ophthalmic preparation for example comprises a sterile, isotonic, buffered to pH 7.4, aqueous solution with gene-regulatory peptide, preferably an NFkappaB-down-regulating peptide as indicated herein at 10-100 microgram/milliliter. Inactive ingredients are for example monobasic and dibasic sodium phosphate, sodium hydroxide to adjust pH, and water for injection.

In another example wherein a gene-regulatory peptide as provided herein is useful as a modulator of NF-kappaB to be used in treatment of disease the invention provides a pharmaceutical composition for systemic application, in particular selected from the group pharmaceutical compositions for intravenous, intraperitoneal, intrathoracal, or intramuscular administration, comprising a gene-regulatory peptide or functional analogue thereof, and use of a gene-regulatory peptide or functional analogue thereof for the production of a pharmaceutical composition for systemic application. Such a composition is most useful to treat the body as a whole and in certain embodiments thereby affects essentially not or only little the area to which it is applied.

In another example wherein a gene-regulatory peptide as provided herein is useful as a modulator of NF-kappaB to be used in treatment of disease, the invention is providing a method and means to treat the systemic immunosuppressive reaction to trauma or surgery by providing a subject believed to be in need thereof with a pharmaceutical composition for systemic application comprising a NF-kappaB down-regulating peptide or functional analogue thereof and an agent directed against disseminated intravascular coagulation. Such an agent may for example be a composition comprising heparin, however, in certain embodiments, the invention provides treatment with a hypotonic pharmaceutical composition for systemic application comprising a NF-kappaB up-regulating peptide or functional analogue thereof. Such treatment may for example comprise infusions with Ringer's lactate for the first 24 hours, the Ringer's lactate provided with, preferably, 1-1000 mg/l NFkappaB regulating peptide such as VLPALPQ (SEQ ID NO:13), GVLPALP (SEQ ID NO:16) or MTRV (SEQ ID NO:20), or mixtures of two or three of these peptides.

During resuscitation, it is often important to keep the volume up, and, if needed, provide the peptide or functional analogue thereof in even further hyopotonic solutions, such as in 0.3 to 0.6% saline. NFkappaB-regulating peptide can be given in the same infusion, the peptide (or analogue) concentration preferably being from about 1 to about 1000 mg/l, but the peptide can also been given in a bolus injection. Doses of 1 to 5 mg/kg bodyweight, for example every eight hours in a bolus injection or per infusionem until the patient stabilizes, are recommended. It is preferred to monitor cytokine profiles, such as TNF-alpha or IL-10 levels, in the plasma of the treated patient, and to stop treatment when these levels are considered within normal boundaries.

In another example wherein a gene-regulatory peptide as provided herein is useful as a modulator of NF-kappaB to be used in treatment of disease, it is herein provided to modulate immunosuppression in a traumatized subject comprising providing the subject with a gene-regulatory peptide comprising a gene-regulatory peptide or functional analogue thereof wherein the subject is also provided with an agent directed against disseminated intravascular coagulation, in particular wherein the agent comprises Activated Protein C activity. Such an agent to modulate disseminated intravascular coagulation (DIC) comprises preferably (recombinant) human Activated Protein C. It is preferably given to the patient per infusionem, whereby NFkappaB regulating peptide can be given in the same infusion, the peptide (or analogue) concentration preferably being from about 1 to about 1000 mg/l, but the peptide can also been given in a bolus injection. Doses of 1 to 5 mg/kg bodyweight, for example every eight hours in a bolus injection or per infusionem until the patient stabilizes, are recommended.

In another example wherein a gene-regulatory peptide as provided herein is useful as a modulator of NF-kappaB to be used in treatment of disease, the invention also provides a hypertonic pharmaceutical composition, such as a resuscitation fluid comprising a NF-kappaB down-regulating peptide or functional analogue thereof. For example, when the subject is in immediate need of a peptide solution to fence of ischemia-reperfusion damage, but time is too short or valuable to give this solution in saline, considering the volume required, it is herein provided to use a hypertonic resuscitation fluid, such as a hypertonic salt solution provided with one or more of the herein mentioned NFkappaB regulatory peptides at a concentration of 1 to 1000 mg/l. For the treatment of trauma or hypovolemic shock in particular various hypertonic pharmaceutical compositions are provided, for example pharmaceutical composition comprising a peptide selected from the group of LQG, AQG, LQGV (SEQ ID NO:1), AQGV (SEQ ID NO:2), LQGA (SEQ ID NO:3), VLPALP (SEQ ID NO:4), ALPALP (SEQ ID NO:5), VAPALP (SEQ ID NO:6), ALPALPQ (SEQ ID NO:7), VLPAAPQ (SEQ ID NO:8), VLPALAQ (SEQ ID NO:9), LAGV (SEQ ID NO:10), VLAALP (SEQ ID NO:11), VLPALA (SEQ ID NO:12), VLPALPQ (SEQ ID NO:13), VLAALPQ (SEQ ID NO:14), VLPALPA (SEQ ID NO:15), GVLPALP (SEQ ID NO:16), LPGC (SEQ ID NO:_, MTRV (SEQ ID NO:20), MTR, and VVC (preferably LAGV (SEQ ID NO:10), AQGV (SEQ ID NO:2), or LQGV (SEQ ID NO:1)). It is particularly preferred to use a hypertonic pharmaceutical composition comprising peptide that is selected from the group of LAGV (SEQ ID NO:10), AQGV (SEQ ID NO:2), and LQGV (SEQ ID NO:1) in cases of SIRS with hypovolemic shock. Furthermore, a method is provided wherein the subject is also provided with an agent directed against disseminated intravascular coagulation, such as wherein the agent comprises Activated Protein C activity.

Administration of such a hypertonic saline (HS) with gene-regulatory peptide to a for example a victim of a traffic accident intravenously causes an initial rapid fluid influx into the vasculature. This is due to the sudden hypertonic state of plasma caused by the infusion of HS (for example, 7.5%, 1283 mmol/l NaCl) in a relatively short time. Other useful sodium concentrations range from 1.2% to 10%. Water is shifted from the intracellular spaces, first the erythrocytes and endothelial cells and then from the tissue cells, into the extracellular compartment. Shrinkage of the endothelium has also beneficial microcirculatory effects due to the reduced resistance of the capillaries. Interstitial water also moves into the intravascular compartment by the osmotic gradient. Hypertonic saline expands intravascular volume by mobilizing fluid that is already present in the body; intracellular and interstitial fluid is shifted into the intravascular space. Plasma volume expansion is therefore with less free water administration than with isotonic plasma expanders. The effect of the hypertonic peptide solution on plasma volume is transient since the fluid will shift from the intravascular space back to the extravascular space, allowing a rapid and thoroughly systemic distribution of a gene-regulatory peptide in the body.

Other circumstances wherein hypertonic solutions as provided herein are useful are for giving immediate,—out-of-hospital—care, such as in an ambulance or at the battlefield, after cardiac attacks and before the patient is transferred to an intensive care unit, and other emergencies where immediate care is needed. Other useful hypertonic solutions may be prepared as well: such as hypertonic NaCl (2400 mosM), hypertonic glucose (2400 mosM), hypertonic sorbitol (2400 mosM), hypertonic glucose (1200 mosM)/glycine (1200 mosM), hypertonic glucose (600 mosM)/mannitol (600 mosM)/glycine (1200 mosM), and hypertonic sorbitol (1200 mosM)/glycine (1200 mosM), each provided with gene-regulatory peptide at 1 to 1000 mg/l. In particular, it is herein provided to use these hypertonic solutions with gene-regulatory peptide initially as a small volume (4-5 ml/kg) infusion fluid. A 80-kg man should receive for example 320-400 ml hypertonic solution with 1-5 mg peptide or functional equivalent/kg body weight. Taking into account an average blood volume of 6 L (6000 ml) and a hematocrit of 45%, the small volume of HS will be distributed into approximately 3300 ml cellular free blood volume. This corresponds to an increase of the cellular free volume of approximately 3620-3700 ml. This also corresponds to approximately 9-11% plasma volume replacement. Initially, for every ml of hypertonic solution with gene regulatory peptide infused, about 7 ml of free water is drawn into the blood stream. Then, once the peptide is distributed and equilibrium is reached, an additional 2,240-2,800 ml free water will be available in the vascular system. This will cause an expansion of the cellular free blood volume, which will reach about 5860-6500 ml. Thus, under equilibrium conditions, approximately 5-6% of the plasma volume will be replaced by hypertonic solution with gene-regulatory peptide. Then, reperfusion of the subject can be continued with normal (i.e., isotonic) reperfusion fluid, such as Ringer's lactate, or even hypotonic solutions of for example hypotonic saline. This results in an expansion of intracellular volume, further facilitating entry of gene-regulatory peptide.

In another example wherein a gene-regulatory peptide as provided herein is useful as a modulator of NF-kappaB to be used in treatment of disease, the invention provides combination therapy that include the concomitant treatment of the patient with a (monoclonal) antibody directed against a cytokine, such as TNF-alpha, IL-6 or IL-12, However, although few would disagree that using these cytokine-blocking agents such as anti-TNF-α therapy may be an important therapeutic addition in the treatment of patients with inflammations such as seen with IBDS, in particular with Crohn's disease, adverse effects related to single cytokine neutralizing therapies have emerged. Also, for unknown reasons, single cytokine blocking proteins may cause the formation of anti-dsDNA antibodies, and after repeated treatment the cumulative ANA incidence can be as high as 50%. Nonetheless, anti-TNF-alpha antibody therapy is associated with lupus-like symptoms. Also, demyelinizing disease and aplastic anemia have been reported in a small number of thus treated patients.

A major problem of repeated administration of chimeric therapeutic antibodies is immunogenicity, and up to 60% of antibody treated patients develop human antichimeric antibodies (HACAs) which are related to infusion reactions and reduce therapeutic efficacy. In comparison with single cytokine therapy, such as the use of anti-TNF-alpha, anti IL-5, anti-IL-6, anti-IL-12, anti-IL-23, anti-IL-12p40, anti-IL23p40 or anti-IL-1beta antibodies, using an NFkappaB down-regulating peptide or functional analogue thereof according to the invention has the major advantage that a major network of pro-inflammatory cytokines is down-regulated.

In another embodiment, it is herein provided to modulate immunosuppression in a traumatized subject comprising providing the subject with a gene-regulatory peptide comprising a gene-regulatory peptide or functional analogue thereof wherein the subject is also provided with an agent directed against disseminated intravascular coagulation, in particular wherein the agent comprises Activated Protein C activity. Such an agent to modulate disseminated intravascular coagulation (DIC) comprises preferably (recombinant) human Activated Protein C. It is preferably given to the patient per infusionem, whereby NFkappaB regulating peptide can be given in the same infusion, the peptide (or analogue) concentration preferably being from about 1 to about 1000 mg/l, but the peptide can also been given in a bolus injection. Doses of 1 to 5 mg/kg bodyweight, for example every eight hours in a bolus injection or per infusionem until the patient stabilizes, are recommended.

In another example wherein a gene-regulatory peptide as provided herein is useful as a modulator of NF-kappaB to be used in treatment of disease, the invention provides use of a NFKB regulating peptide or derivative thereof for the production of a pharmaceutical composition for the treatment of inflammatory disease in a primate, and provides a method of treatment of inflammatory disease in a primate. It is preferred that the treatment comprises administering to the subject a pharmaceutical composition comprising an NFkappaB down-regulating peptide or functional analogue thereof. Examples of useful NFkappaB down-regulating peptides are VLPALPQVVC (SEQ ID NO:_), LQGVLPALPQ (SEQ ID NO:_), LQG, LQGV (SEQ ID NO:1), GVLPALPQ (SEQ ID NO:16), VLPALP (SEQ ID NO:4), VVC, MTR and circular LQGVLPALPQVVC (SEQ ID NO:17). More down-regulating peptides and functional analogues can be found using the methods as provided herein. Most prominent among NFkappaB down-regulating peptides are VLPALPQVVC (SEQ ID NO:_), LQGVLPALPQ (SEQ ID NO:_), LQG, LQGV (SEQ ID NO:1), and VLPALP (SEQ ID NO:4). These are also capable of reducing production of NO by a cell.

In one embodiment, the invention provides a method of treating a subject suffering from an inflammatory disease with a method and gene-regulatory peptide according to the invention concomitantly, or at least timely, with a treatment with a single cytokine blocking protein, such as an anti-TNF-alpha, anti IL-5, anti-IL-6, anti-IL-12, anti-IL-23, anti-IL-12p40, anti-IL23p40 or anti-IL-1beta antibody or functional analogue thereof.

It is herein also provided to use a gene-regulatory peptide according to the invention for the production of a pharmaceutical composition for the treatment of a subject believed to be suffering of an inflammation and receiving treatment with an anti-cytokine antibody such as an anti-TNF-alpha, anti IL-5, anti-IL-6, anti-IL-12, anti-IL-23, anti-IL-12p40, anti-IL23p40 or anti-IL-1beta antibody.

It is herein also provided to use a composition that comprises at least two oligopeptides or functional analogues thereof, each capable of down-regulation NFkappaB, and thereby reducing production of NO and/or TNF-alpha by a cell, in particular wherein at least two oligopeptides are selected from the group LQGV (SEQ ID NO:1), AQGV (SEQ ID NO:2) and VLPALP (SEQ ID NO:4), for the treatment of inflammatory diseased patients that are also treated with an anti-cytokine antibody. In a particular example wherein a gene-regulatory peptide as provided herein is useful as a modulator of NF-kappaB to be used in treatment of disease, such treatment as provided herein is applicable to patients suffering from inflammatory bowels disease (IBD). Inflammatory bowel disease (IBD) is a chronic relapsing and remitting inflammatory condition of the gastrointestinal tract that is manifest as 1 of 2 usually distinct but sometimes overlapping clinical entities, ulcerative colitis (UC) and Crohn's disease (CD). Ulcerative colitis affects the colon and is a superficial ulcerative disease, whereas CD is a transmural granulomatous disorder that affects any part of the gastrointestinal tract and has a predilection for the terminal ileum and colon. Both forms of IBD are associated with prominent extra-intestinal manifestations and an increased incidence of gastrointestinal cancer, in addition, both begin relatively early in life and persist for long periods, leading to decreased quality of life indices and a greater than 2-fold increase in mortality rate.

By and large, IBD is a disease of “urbanized” areas such as the United States and Europe, where it occurs at an incidence of 6 to 12 and 5 to 7 per 100,000 population for UC and CD, respectively. This translates to 45,000 new cases per year and one million affected individuals in the United States alone and costs the US health care system approximately $1.8 billion per year (1990 estimate).

The concept that the proximal cause of IBD is immunologic in nature arose from the observation that IBD is characterized by massive cellular infiltrates and is associated with abnormalities of the immune system that include NFkappaB induced inappropriate production of antibodies and T-cell dysfunctions. This concept has been clarified by studies of patient lamina propria (LP) cells that show that in Crohn's disease (CD), NFkappaB induces immune cells to overproduce cytokines indicative of a typical helper T-cell 1 (T_(H)1) response, namely increased production of interleukin (IL) 12 by LP macrophages and increase production of interferon (IFN) γ by LP T cells. In addition, LP T cells from patients with ulcerative colitis (UC) manifest a cytokine profile compatible with a T_(H)2 response; thus, while the cells do not overproduce the major T_(H)2 cytokine IL-4, they do produce increased amounts of another T_(H)2 cytokine, IL-5. This cytokine production pattern accords with an association of UC (but not CD) with auto-antibodies that in general require T_(H)2 responses.

These data provide evidence that the 2 major forms of IBD are due to dysregulated or excessive T_(H)1 (CD) or T_(H)2 (UC) responses. As to what factors induce these abnormal responses, there is considerable evidence that IBD patients have inappropriate T-cell responses to antigenic components of their own intestinal microflora, that benefit from treatment with a gene-regulatory peptide as provided herein to serve as a modulator of NF-kappaB to be used in treatment of IBD both because of the beneficial effects on the earlier dysfunction in the primary or secondary mechanisms that normally drive and regulate such response and because of down-regulation of the inflammatory cascade in the intestinal epithelial cell barrier that otherwise leads to inappropriate penetration of microbial antigens.

In another example wherein a gene-regulatory peptide as provided herein is useful as a modulator of NF-kappaB to be used in treatment of disease, the invention provides treatment with a gene-regulatory peptide concomitantly with selective decontamination of the gut, as practiced in some patients, for example in preparation of a patient for a bone marrow transplantation, induces additional pro-inflammatory cytokine release, which can add to the pro-inflammatory burst in case of a complication such as hemorrhagic shock. Selective decontamination of the gut, as practiced in some patients, for example in preparation of a bone marrow transplant, induces additional proinflammatory cytokine release, which can add to the proinflammatory burst in case of a complication such as hemorrhagic shock. Also, major surgery, such as cardiopulmonary bypass predisposes the splanchnic region to inadequate perfusion and increases in gut permeability.

Related to these changes, circulating endotoxin has been shown to rise during surgery, and contributes to cytokine activation, high oxygen consumption, and fever (“post-perfusion syndrome”). To a large extent, free endotoxin in the gut is a product of the proliferation of aerobic gram-negative bacteria and may be reduced by nonabsorbable antibiotics, however, selective decontamination of the gut does not affect the occurrence of perioperative endotoxemia, nor does it reduce the tumor necrosis factor-alpha or interleukin-6 concentrations as determined before surgery, upon aorta declamping, 30 minutes into reperfusion, or two hours after surgery. Also, selective decontamination of the gut does not alter the incidence of postoperative fever or clinical outcome measures such as duration of artificial ventilation and intensive care unit and hospital stay. In conclusion, treatment with a gene-regulatory peptide as provided herein is useful as a modulator of NF-kappaB to affect the incidence of perioperative endotoxemia, pro-inflammatory cytokine activation and the occurrence of a post-perfusion syndrome during or after surgery.

In another example wherein a gene-regulatory peptide as provided herein is useful as a modulator of NF-kappaB to be used in treatment of disease, the invention provides a pharmaceutical composition for the treatment of an overly strong immune response occurring in a primate, and a method for the treatment of an overly strong immune response resulting in additional pro-inflammatory cytokine release in a primate comprising subjecting the subject to a gene-regulatory peptide according to the invention, preferably to a mixture of such gene-regulatory peptides. Administration of such a gene-regulatory peptide or mixture preferably occurs systemically, e.g., by intravenous or intraperitoneal administration and leads to a dampening of the effect of the additionally released pro-inflammatory cytokines. In a further embodiment, such treatment also comprises the use of for example an antimicrobial agent, however, especially when such use is otherwise contraindicated or at least considered at risk because of the chance of generating toxin loads that lead to an additional pro-inflammatory cytokine response because of lysis of the microbe subject to the action of those antibiotics in an individual thus treated.

In another example wherein a gene-regulatory peptide as provided herein is useful as a modulator of NF-kappaB to be used in treatment of disease, the invention provides a mode of treatment of the additional pro-inflammatory cytokine response seen after surgical interventions.

The invention also provides a method for treating a subject that is or has already been treated with another pharmaceutical composition and believed to be suffering or at risk from the side-effects of such a composition or at least believed to profit from the here provided concomitant therapy comprising providing the subject with a gene-regulatory peptide comprising a short, gene regulatory peptide or functional analogue thereof, wherein the gene-regulatory peptide is administered in an amount sufficient to modulate possible side-effects, for example wherein the another pharmaceutical composition is selected from the group of antigens, vaccines, antibodies, anticoagulants, antibiotics, in particular beta-lactam antibiotics, antitoxins, antibacterial agents, antiparasitic agents, antiprotozootic agents, antifungal agents, antiviral agents, cytolytic agents, cytostatic agents, thrombolytic agents, and others. In certain embodiments, the invention provides a method of treating a subject with a method and gene-regulatory peptide according to the invention concomitantly, or at least timely, with an thrombolytic agent, such as (recombinant) tissue plasminogen activator, or truncated forms thereof having tissue plasminogen activity, or streptokinase, or urokinase.

Also provided is a method for modulating an iatrogenic event in a subject that is concomitantly or already treated with a pharmaceutical composition and believed to be suffering or at risk from the side-effects of such a composition comprising providing the subject with a gene-regulatory peptide comprising a short, gene regulatory peptide or functional analogue thereof, wherein the gene-regulatory peptide is administered in an amount sufficient to modulate the resulting inflammatory cascade, for example wherein the pharmaceutical composition is selected from the group of antigens, vaccines, antibodies, anticoagulants, antibiotics, in particular beta-lactam antibiotics, antitoxins, antibacterial agents, antiparasitic agents, antiprotozootic agents, antifungal agents, antiviral agents, cytolytic agents, cytostatic agents, thrombolytic agents. Provided is, for example, a method to control the toxic effects of the lysis or damage to bacterial pathogens which release endotoxin and a host of other enterotoxin and exotoxins, resulting in an at times undesirable pro-inflammatory cytokine cascade. Furthermore, provided is a method to treat a viral infection.

Also provided is a method wherein the iatrogenic event includes the treatment of a subject with a virus, especially wherein lysis is due to treatment of the subject with the virus. A clear example of the beneficial use of a peptide or functional analogue thereof according to the invention to control a therapy-impeding inflammatory reaction relates to the example of an inflammatory response to (for example adenoviral or retroviral) gene vectors, e.g., in gene therapy such as in treatment of cystic fibrosis. The peptides can be administered systemically as indicated above in the case of cystic fibrosis gene therapy. In another example, the virus comprises a lytic phage used in antibacterial therapy as discussed above.

In another example wherein a gene-regulatory peptide as provided herein is useful as a modulator of NF-kappaB to be used in treatment of disease, a peptide according to the invention, or a functional derivative or analogue thereof is used for the production of a pharmaceutical composition, for the treatment or mitigation of a pro-inflammatory cytokine response seen after a virus infection. Examples of useful NFkappaB down-regulating peptides to be included in such a pharmaceutical composition are VLPALPQVVC (SEQ ID NO:_), LQGVLPALPQ (SEQ ID NO:_), LQG, LQGV (SEQ ID NO:1), GVLPALPQ (SEQ ID NO:16), VLPALP (SEQ ID NO:4), VVC, MTR and circular LQGVLPALPQVVC (SEQ ID NO:17), which are for example useful in the treatment of an overly strong IL-6 response seen after severe acute respiratory syndrome (SARS). It is preferred to include concomitant treatment with an antiviral agent such as ribavirine and a gene-regulatory peptide to reduce for example NFkappaB modulated IL-6 activity as provided herein.

More gene-regulating peptides and functional analogues can be found in a (bio)assay, such as a NFkappaB translocation assay as provided herein. Most prominent among NFkappaB down-regulating peptides are VLPALPQVVC (SEQ ID NO:_), LQGVLPALPQ (SEQ ID NO:_), LQG, LQGV (SEQ ID NO:1), and VLPALP. These are also capable of reducing production of NO by a cell. It is herein also provided to use a composition that comprises at least two oligopeptides or functional analogues thereof, each capable of down-regulation NFkappaB, and thereby reducing production of NO and/or TNF-alpha by a cell, in particular wherein the at least two oligopeptides are selected from the group LQGV (SEQ ID NO:1), AQGV and VLPALP (SEQ ID NO:4). Useful NFkappaB up-regulating peptides are VLPALPQ (SEQ ID NO:13), GVLPALP (SEQ ID NO:16), and MTRV (SEQ ID NO:20). As indicated, more gene-regulatory peptides may be founds with an appropriate assay or bioassay. A gene-regulatory peptide as used herein is preferably short. Preferably, such a peptide is 3 to 15 amino acids long, and capable of modulating the expression of a gene, such as a cytokine, in a cell.

In certain embodiments, a peptide is a gene-regulatory peptide that is capable of traversing the plasma membrane of a cell or, in other words, a peptide that is membrane-permeable, more preferably, wherein the lead peptide is three to nine amino acids long, most preferred wherein the gene-regulatory peptide is four to six amino acids long.

In certain embodiments, a gene-regulatory peptide is administered in an effective concentration to an animal or human systemically, e.g., by intravenous, intra-muscular or intraperitoneal administration. Another way of administration comprises perfusion of organs or tissue, be it in vivo or ex vivo, with a perfusion fluid comprising a gene-regulatory peptide according to the invention.

In patients with contusio cerebri and intracranial pressure treatment it is advantageous to combine treatment with the peptides or functional analogues thereof with osmotic agents like mannitol to reduce intracranial pressure and stimulate cerebral perfusion, i.e., by administering intravenous infusions of mannitol 20% in 0.9% saline solutions of 200 ml, or another hypertonic solution, one to six times a day. NFkappaB regulating peptide can be given in the same infusion, the peptide (or analogue) concentration preferably being from about 1 to about 1000 mg/l, but the peptide can also been given in a bolus injection. Doses of 1 to 5 mg/kg bodyweight, for example every eight hours in a bolus injection or per infusionem until the patient stabilizes, are recommended.

In the case of a cardiovascular or cerebrovascular incident, such treatment can for example take the form of intravenous infusions of recombinant tissue plasminogen activator (rt-PA) at a dose of 0.9 mg/kg (maximum of 90 mg) in 0.9% saline solutions, whereby it is preferred that 10% of the rt-PA dose is given within one to two minutes and the remaining dose of rt-PA in 60 minutes. In the case of an acute myocardial infarction, such treatment can for example take the form of intravenous infusions of rt-PA at a dose of 15 mg as intravenous bolus, followed by 50 mg in the next 30 minutes followed by 35 mg in the next 60 minutes. For the sake of treating the resulting perfusion injury that occurs due to the lysis of the thrombus and the subsequent perfusion of the ischemic area, it is herein provided to also provide the patient with a bolus injection of NF-kappaB down-regulating peptide such as AQGV (SEQ ID NO:2), LQGV or VLPALP at 2 mg/kg and continue the infusion with a NF-kappaB down-regulating peptide such as AQGV (SEQ ID NO:2), LQGV (SEQ ID NO:1), or VLPALP (SEQ ID NO:4) or a functional analogue thereof at a dose of 1 mg/kg bodyweight for every eight hours.

Also of clinical and medical interest and value, the present invention provides the opportunity to selectively control NFKB-dependent gene expression in tissues and organs in a living subject, preferably in a primate, allowing up-regulating essentially anti-inflammatory responses such as IL-10, and down-regulating essentially pro-inflammatory responses such as mediated by TNF-alpha, nitric oxide (NO), IL-5, IL-6, IL-12 and IL-1beta. For example, in comparison with single cytokine therapy, such as the use of anti-TNF-alpha, anti IL-5, anti-IL-6, anti-IL-12 or anti-IL-1beta antibodies, using a NFkappaB down-regulating peptide or functional analogue thereof according to the invention has the distinct advantage that a major network of pro-inflammatory cytokines is down-regulated.

It is preferred when the treatment comprises administering to the subject a pharmaceutical composition comprising an NFkappaB down-regulating peptide or functional analogue thereof. Examples of useful NFkappaB down-regulating peptides are VLPALPQVVC (SEQ ID NO:_), LQGVLPALPQ (SEQ ID NO:_), LQG, LQGV (SEQ ID NO:1), GVLPALPQ (SEQ ID NO:16), VLPALP (SEQ ID NO:4), VVC, MTR and circular LQGVLPALPQVVC (SEQ ID NO:_). More down-regulating peptides and functional analogues can be found using the methods as provided herein. Most prominent among NFkappaB down-regulating peptides are VLPALPQVVC (SEQ ID NO:_), LQGVLPALPQ (SEQ ID NO:_), LQG, LQGV (SEQ ID NO:1), and VLPALP (SEQ ID NO:4). These are also capable of reducing production of NO by a cell. In one embodiment, the invention provides a method of treating a subject suffering from a relapsing/remitting disease seen with MS with a method and gene-regulatory peptide according to the invention concomitantly, or at least timely, with a treatment with a single cytokine blocking protein, such as an anti-TNF-alpha, anti IL-5, anti-IL-6, anti-IL-12 or anti-IL-1beta antibody or functional analogue thereof.

It is herein also provided to use a gene-regulatory peptide according to the invention for the production of a pharmaceutical composition for the treatment of a subject believed to be suffering of an inflammation, such as Crohn's disease or multiple sclerosis and receiving treatment with an anti-TNF-alpha, anti IL-5, anti-IL-6, anti-IL-12 or anti-IL-1beta antibody. It is herein also provided to use a composition that comprises at least two oligopeptides or functional analogues thereof, each capable of down-regulation NFkappaB, and thereby reducing production of NO and/or TNF-alpha by a cell, in particular wherein the at least two oligopeptides are selected from the group LQGV (SEQ ID NO:1), AQGV (SEQ ID NO:2), and VLPALP (SEQ ID NO:4), for the treatment of for the treatment of a subject believed to be suffering of an inflammation, such as Crohn's disease or multiple sclerosis and receiving treatment with an anti-TNF-alpha, anti IL-5, anti-IL-6, anti-IL-12 or anti-IL-1beta antibody.

The invention provides a method for modulating transplant survival in a recipient of the transplant comprising providing the transplant with a gene-regulatory peptide comprising a peptide or functional analogue thereof. It is preferred that the peptide is three to 15 amino acids long, more preferably, that the peptide is three to nine amino acids long, it most preferred that the peptide is four to six amino acids long. It is in particular preferred that the gene-regulatory peptide is capable of inhibiting NF-kappaB/Rel protein activity. Functional analogue herein relates to the signaling molecular effect or activity as for example can be measured by measuring nuclear translocation of a relevant transcription factor, such as NF-kappaB in an NF-kappaB assay, or AP-1 in an AP-1 assay, or by another method as provided herein. Fragments can be somewhat (i.e., one or two amino acids) smaller or larger on one or both sides, while still providing functional activity.

In one embodiment of the invention, the peptide used as a gene-regulatory peptide a chemically modified peptide. A peptide modification includes phosphorylation (e.g., on a Tyr, Ser or Thr residue), N-terminal acetylation, C-terminal amidation, C-terminal hydrazide, C-terminal methyl ester, fatty acid attachment, sulfonation (tyrosine), N-terminal dansylation, N-terminal succinylation, tripalmitoyl-S-Glyceryl Cysteine (PAMb 3 Cys-OH) as well as famesylation of a Cys residue. Systematic chemical modification of a peptide can, for example, be performed in the process of peptide optimalization.

Synthetic peptides can be obtained using various procedures known in the art. These include solid phase peptide synthesis (SPPS) and solution phase organic synthesis (SPOS) technologies. SPPS is a quick and easy approach to synthesize peptides and small proteins. The C-terminal amino acid is typically attached to a cross-linked polystyrene resin via an acid labile bond with a linker molecule. This resin is insoluble in the solvents used for synthesis, making it relatively simple and fast to wash away excess reagents and by-products. The peptide, or its functional analogue, modification or derivative, can be administered as the entity as such or as a pharmaceutically acceptable acid- or base-addition salt, formed by reaction with an inorganic acid (such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid); or with an organic acid (such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid); or by reaction with an inorganic base (such as sodium hydroxide, ammonium hydroxide, potassium hydroxide); or with an organic base (such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines). A selected peptide and any of the derived entities may also be conjugated to sugars, lipids, other polypeptides, nucleic acids and PNA; and function in-situ as a conjugate or be released locally after reaching a targeted tissue or organ.

In response to a variety of pathophysiological and developmental signals, the NFkB/Rel family of transcription factors are activated and form different types of hetero- and homodimers among themselves to regulate the expression of target genes containing kappaB-specific binding sites. NF-KB transcription factors are hetero- or homodimers of a family of related proteins characterized by the Rel homology domain. They form two subfamilies, those containing activation domains (p65-RELA, RELB, and c-REL) and those lacking activation domains (p50, p52). The prototypical NFkB is a heterodimer of p65 (RELA) and p50 (NF-kB1). Among the activated NFkB dimers, p50-p65 heterodimers are known to be involved in enhancing the transcription of target genes and p50-p50 homodimers in transcriptional repression. However, p65-p65 homodimers are known for both transcriptional activation and repressive activity against target genes. KappaB DNA binding sites with varied affinities to different NFB dimers have been discovered in the promoters of several eukaryotic genes and the balance between activated NFkB homo- and heterodimers ultimately determines the nature and level of gene expression within the cell.

The term “NFkB-regulating peptide” as used herein refers to a peptide or functional analogue or a modification or derivative thereof capable of modulating the activation of members of the NFkB/Rel family of transcription factors. Activation of NFkB can lead to enhanced transcription of target genes. Also, it can lead to transcriptional repression of target genes. NFkB activation can be regulated at multiple levels. For example, the dynamic shuttling of the inactive NFkB dimers between the cytoplasm and nucleus by IkappaB proteins and its termination by phosphorylation and proteasomal degradation, direct phosphorylation, acetylation of NFkB factors, and dynamic reorganization of NFkB subunits among the activated NFkB dimers have all been identified as key regulatory steps in NFkB activation and, consequently, in NFkB-mediated transcription processes. Thus, an NFkB-regulating peptide is capable of modulating the transcription of genes that are under the control of NFkB/Rel family of transcription factors. Modulating comprises the up-regulation or the down-regulation of transcription.

The term “pharmaceutical composition” as used herein is intended to cover both the active gene-regulatory peptide alone or a composition containing the gene-regulatory peptide together with a pharmaceutically acceptable carrier, diluent or excipient. Acceptable diluents of an oligopeptide as described herein in the detailed description are for example physiological salt solutions or phosphate buffered salt solutions.

In response to a variety of pathophysiological and developmental signals, the NFkB/Rel family of transcription factors are activated and form different types of hetero- and homodimers among themselves to regulate the expression of target genes containing kappaB-specific binding sites. NF-kB transcription factors are hetero- or homodimers of a family of related proteins characterized by the Rel homology domain. They form two subfamilies, those containing activation domains (p65-RELA, RELB, and c-REL) and those lacking activation domains (p50, p52). The prototypical NFkB is a heterodimer of p65 (RELA) and p50 (NF-kB1). Among the activated NFkB dimers, p50-p65 heterodimers are known to be involved in enhancing the transcription of target genes and p50-p50 homodimers in transcriptional repression. However, p65-p65 homodimers are known for both transcriptional activation and repressive activity against target genes.

KappaB DNA binding sites with varied affinities to different NFkappaB dimers have been discovered in the promoters of several eukaryotic genes and the balance between activated NFkB homo- and heterodimers ultimately determines the nature and level of gene expression within the cell. The term “NFkB-regulating peptide” as used herein refers to a peptide or a modification or derivative thereof capable of modulating the activation of members of the NFkB/Rel family of transcription factors. Activation of NFkB can lead to enhanced transcription of target genes. Also, it can lead to transcriptional repression of target genes. NFkB activation can be regulated at multiple levels. For example, the dynamic shuttling of the inactive NFkB dimers between the cytoplasm and nucleus by IkappaB proteins and its termination by phosphorylation and proteasomal degradation, direct phosphorylation, acetylation of NFkB factors, and dynamic reorganization of NFkB subunits among the activated NFkB dimers have all been identified as key regulatory steps in NFkB activation and, consequently, in NFkB-mediated transcription processes. Thus, an NFkB-regulating peptide is capable of modulating the transcription of genes that are under the control of the NFkB/Rel family of transcription factors. Modulation comprises the up-regulation or the down-regulation of transcription.

In certain embodiments, a peptide or a functional derivative or analogue thereof is used for the production of a pharmaceutical composition for the treatment of a parasitic infection or the treatment of an inflammatory condition found after a parasitic infection, such as seen with malaria.

Use of a gene-regulatory peptide or analogue thereof is herein also provided to control many of the inflammatory, allergic and other immune-mediated problems that are caused by parasitic diseases, such as malaria, onchocerciasis, lymphatic filariasis, and trachoma, among others. These inflammatory problems may occur in patients even when they have been successfully treated for the underlying parasitic infection, because the inflammatory response can persist for long periods of time after the parasites have been eliminated. Of course, the need for an anti-inflammatory peptide product is even greater when patients have been infected with parasites that are partially or fully resistant to available anti-parasitic drugs.

The World Health Organization (“WHO”) states that there are an estimated 500 million people in the world who have malaria, with two million deaths each year from the disease—mostly in younger children. Despite strenuous efforts on the part of WHO and other institutions, the disease has been very difficult to control, because mosquitoes become resistant to the insecticides, parasites become resistant to the anti-malarial agents and the inflammatory and immune problems persist even after the parasites are cleared.

Although a gene-regulatory peptide is not expected to kill the parasites that devastate hundreds of millions of people in the developing world, they mediate both (a) the acute, life threatening cytokine-induced inflammatory conditions, and (b) the sub-acute highly debilitating auto-immune and allergic-type reactions, that are associated with many of these parasitic diseases. The parasitic diseases (notably malaria, and especially in malaria caused by P. falciparum) tend to induce cytokines and other immune mediators (such as nitric oxide), triggering SIRS, and resulting in multi-organ dysfunction syndrome (“MODS”), disseminated intravascular coagulation (“DIC”), and multi-organ failure (“MOF”). In other words, many parasites trigger the same reactions from the immune system (i.e., trigger the release of the same mediators) that are triggered by bacterial infections, and can lead to SIRS. Using gene-regulatory peptide therapy as provided herein will significantly slow down the various types of cytokine-driven disease progression—even in those cases where the parasite is susceptible to anti-infective agents, including and especially cerebral malaria.

Sub-acute auto-immune and allergic-type reactions are also induced by parasites. For example, malaria demonstrates the dangerous effects of the auto-immune reactions that can occur (and persist) even when anti-malarial agents have succeeded in clearing the tissues—in this case, red blood cells (“RBCs”)—of parasites. It is not uncommon for millions of RBCs that were never infected by parasites to be destroyed. This hemolysis is due to auto-antibodies and other immune factors, which induce apoptosis (cell death). Thus, even when the parasites have been eradicated by antimalarial drugs, patients often develop anemia that is life-threatening. The threat to life is due not only to the profound anemia, but also to the damage caused to the kidneys by the significant quantities of intracellular contents that develop upon the lysis of vast numbers of RBCs. This problem is particularly acute with respect to the “Blackwater fever” variant of malaria, where the mortality is 20-30%, due to massive intravascular hemolysis leading to acute renal failure, even though parasitemia is absent at the time that hemolysis occurs. Use of an inflammatory peptide product according to the invention affords significant reduction in this rate of mortality.

In many of the parasitic diseases, there are repeat infections over the course of years. Eventually individuals may build up sufficient immunity that the subsequent infections decrease in severity. However, over the course of these repeat infections the intense allergic reactions (to various components of the infecting organism) cause a great deal of tissue destruction. These allergic reactions, which are IgE-mediated, can cause the deposition of IgE-containing immune complexes in (i) small blood vessels, leading to overproduction of TNF-a with all its complications, (e.g., plugging of the blood vessels), and (ii) in the conjuctiva of the eyes, leading to scarring and blindness.

There are several types of interactions between IgE and transcription factor NF-kappaB (also called NF-κB). The interactions between NF-κB and IgE include the following:

-   -   IgE isotype switching is dependent on the binding of NF-kB to a         gene promoter.     -   Aggregation of the high-affinity IgE receptor on monocytes and         dendritic cells leads to synthesis and release of TNF-A and         monocyte chemoattractant protein-1.

The parasite products, like endotoxin, induce activation of the cytokine cascade. Cytokine production may also be stimulated by IgE-antigen or IgE-antilgE complexes. Thus, parasite product and antibody complexes stimulate cells of the macrophage-monocyte series, and possibly endothelium, to release inflammatory cytokines in malaria.

Using a gene-regulatory peptide that is capable of down-regulating inflammatory pathways as provided herein will reduce the extent of the IgE-induced allergic damage and other systemic inflammatory conditions that takes place in many of the chronic/recurring parasitic diseases. Examples of useful NFkappaB down-regulating peptides to be included in such a pharmaceutical composition are VLPALPQVVC (SEQ ID NO:_), LQGVLPALPQ (SEQ ID NO:_), LQG, LQGV (SEQ ID NO:1), GVLPALPQ (SEQ ID NO:16), VLPALP (SEQ ID NO:4), VVC, MTR and circular LQGVLPALPQVVC (SEQ ID NO:17). More gene-regulating peptides and functional analogues can be found in a (bio)assay, such as a NFkappaB translocation assay as provided herein. Most prominent among NFkappaB down-regulating peptides are VLPALPQVVC (SEQ ID NO:_), LQGVLPALPQ (SEQ ID NO:_), LQG, LQGV (SEQ ID NO:1), and VLPALP (SEQ ID NO:4). These are also capable of reducing production of NO by a cell. Furthermore, LQG, VVC and MTRV (SEQ ID NO:20), and, in particular, LQGV (SEQ ID NO:1) promote angiogenesis, especially in topical applications.

It is herein also provided to use a composition that comprises at least two oligopeptides or functional analogues thereof, each capable of down-regulation NFkappaB, and thereby reducing production of NO and/or TNF-alpha by a cell, in particular wherein the at least two oligopeptides are selected from the group LQGV (SEQ ID NO:1), AQGV (SEQ ID NO:2) and VLPALP (SEQ ID NO:4). Useful NFkappaB up-regulating peptides are VLPALPQ (SEQ ID NO:13), GVLPALP (SEQ ID NO:16), and MTRV (SEQ ID NO:20). As indicated, more gene-regulatory peptides may be found with an appropriate (bio)assay. A gene-regulatory peptide as used herein is preferably short. Preferably, such a peptide is three to 15 amino acids long, more preferably, wherein the lead peptide is three to nine amino acids long, most preferred wherein the lead peptide is four to six amino acids long, and capable of modulating the expression of a gene, such as a cytokine, in a cell.

In certain embodiments, a peptide is a gene-regulatory peptide that is capable of traversing the plasma membrane of a cell or, in other words, a peptide that is membrane-permeable.

Functional derivative or analogue herein relates to the signaling molecular effect or activity as for example can be measured by measuring nuclear translocation of a relevant transcription factor, such as NF-kappaB in an NF-kappaB assay, or AP-1 in an AP-1 assay, or by another method as provided herein. Fragments can be somewhat (i.e., one or two amino acids) smaller or larger on one or both sides, while still providing functional activity. Such a bioassay comprises an assay for obtaining information about the capacity or tendency of a peptide, or a modification thereof, to regulate expression of a gene. A scan with, for example, a 15-mer, or a 12-mer, or a 9-mer, or a 8-mer, or a 7-mer, or a 6-mer, or a 5-mer, or a 4-mer or a 3-mer peptides can yield valuable information on the linear stretch of amino acids that form an interaction site and allows identification of gene-regulatory peptides that have the capacity or tendency to regulate gene expression. Gene-regulatory peptides can be modified to modulate their capacity or tendency to regulate gene expression, which can be easily assayed in an in vitro bioassay such as a reporter assay. For example, a particular amino acid at an individual position can be replaced with another amino acid of similar or different properties.

Alanine (Ala)-replacement scanning, involving a systematic replacement of each amino acid by an Ala residue, is a suitable approach to modify the amino acid composition of a gene-regulatory peptide when in a search for a gene-regulatory peptide capable of modulating gene expression. Of course, such replacement scanning or mapping can be undertaken with amino acids other than Ala as well, for example with D-amino acids. In one embodiment, a peptide derived from a naturally occurring polypeptide is identified as being capable of modulating gene expression of a gene in a cell. Subsequently, various synthetic Ala-mutants of this gene-regulatory peptide are produced. These Ala-mutants are screened for their enhanced or improved capacity to regulate expression of a gene compared to gene-regulatory polypeptide.

Furthermore, a gene-regulatory peptide, or a modification or analogue thereof, can be chemically synthesized using D- and/or L-stereoisomers. For example, a gene-regulatory peptide that is a retro-inverso of an oligopeptide of natural origin is produced. The concept of polypeptide retro-inversion (assembly of a natural L-amino acid-containing parent sequence in reverse order using D-amino acids) has been applied successfully to synthetic peptides. Retro-inverso modification of peptide bonds has evolved into a widely used peptidomimetic approach for the design of novel bioactive molecules which has been applied to many families of biologically active peptides. The sequence, amino acid composition and length of a peptide will influence whether correct assembly and purification are feasible. These factors also determine the solubility of the final product.

The purity of a crude peptide typically decreases as the length increases. The yield of peptide for sequences less than 15 residues is usually satisfactory, and such peptides can typically be made without difficulty. The overall amino acid composition of a peptide is an important design variable. A peptide's solubility is strongly influenced by composition. Peptides with a high content of hydrophobic residues, such as Leu, Val, Ile, Met, Phe and Trp, will either have limited solubility in aqueous solution or be completely insoluble. Under these conditions, it can be difficult to use the peptide in experiments, and it may be difficult to purify the peptide if necessary. To achieve a good solubility, it is advisable to keep the hydrophobic amino acid content below 50% and to make sure that there is at least one charged residue for every five amino acids. At physiological pH Asp, Glu, Lys, and Arg all have charged side chains. A single conservative replacement, such as replacing Ala with Gly, or adding a set of polar residues to the N- or C-terminus, may also improve solubility.

Peptides containing multiple Cys, Met, or Trp residues can also be difficult to obtain in high purity partly because these residues are susceptible to oxidation and/or side reactions. If possible, one should choose sequences to minimize these residues. Alternatively, conservative replacements can be made for some residues. For instance, Norleucine can be used as a replacement for Met, and Ser is sometimes used as a less reactive replacement for Cys. If a number of sequential or overlapping peptides from a protein sequence are to be made, making a change in the starting point of each peptide may create a better balance between hydrophilic and hydrophobic residues. A change in the number of Cys, Met, and Trp residues contained in individual peptides may produce a similar effect.

In another embodiment of the invention, a gene-regulatory peptide capable of modulating gene expression is a chemically modified peptide. A peptide modification includes phosphorylation (e.g., on a Tyr, Ser or Thr residue), N-terminal acetylation, C-terminal amidation, C-terminal hydrazide, C-terminal methyl ester, fatty acid attachment, sulfonation (tyrosine), N-terminal dansylation, N-terminal succinylation, tripalmitoyl-S-Glyceryl Cysteine (PAM3 Cys-OH) as well as famesylation of a Cys residue. Systematic chemical modification of a gene-regulatory peptide can, for example, be performed in the process of gene-regulatory peptide optimization.

Synthetic peptides can be obtained using various procedures known in the art. These include solid phase peptide synthesis (SPPS) and solution phase organic synthesis (SPOS) technologies. SPPS is a quick and easy approach to synthesize peptides and small proteins. The C-terminal amino acid is typically attached to a cross-linked polystyrene resin via an acid labile bond with a linker molecule. This resin is insoluble in the solvents used for synthesis, making it relatively simple and fast to wash away excess reagents and by-products.

The peptides as mentioned in this document such as LQG, AQG, LQGV (SEQ ID NO:1), AQGV (SEQ ID NO:2), LQGA (SEQ ID NO:3), VLPALP (SEQ ID NO:4), ALPALP (SEQ ID NO:5), VAPALP (SEQ ID NO:6), ALPALPQ (SEQ ID NO:7), VLPAAPQ (SEQ ID NO:8), VLPALAQ (SEQ ID NO:9), LAGV (SEQ ID NO:10), VLAALP (SEQ ID NO:11), VLPALA (SEQ ID NO:12), VLPALPQ (SEQ ID NO:13), VLAALPQ (SEQ ID NO:14), VLPALPA (SEQ ID NO:15), GVLPALP (SEQ ID NO:16), VVCNYRDVRFESIRLPGCPRGVNPVVSYAVALSCQCAL (SEQ ID NO:24), RPRCRPINATLAVEKEGCPVCITVNTTICAGYCPT (SEQ ID NO:25), SKAPPPSLPSPSRLPGPS (SEQ ID NO:26), LQGVLPALPQVVC (SEQ ID NO:17), SIRLPGCPRGVNPVVS (SEQ ID NO:27), LPGCPRGVNPVVS (SEQ ID NO:18), LPGC (SEQ ID NO:_), MTRV (SEQ ID NO:20), MTR, and VVC were prepared by solid-phase synthesis using the fluorenylmethoxycarbonyl (Fmoc)/tert-butyl-based methodology with 2-chlorotrityl chloride resin as the solid support. The side-chain of glutamine was protected with a trityl function. The peptides were synthesized manually. Each coupling consisted of the following steps: (i) removal of the alpha-amino Fmoc-protection by piperidine in dimethylformamide (DMF), (ii) coupling of the Fmoc amino acid (3 eq) with diisopropylcarbodiimide (DIC)/1-hydroxybenzotriazole (HOBt) in DMF/N-methylformamide (NMP) and (iii) capping of the remaining amino functions with acetic anhydride/diisopropylethylamine (DIEA) in DMF/NMP. Upon completion of the synthesis, the peptide resin was treated with a mixture of trifluoroacetic acid (TFA)/H₂O/triisopropylsilane (TIS) 95:2.5:2.5. After 30 minutes, TIS was added until decolorization. The solution was evaporated in vacuo and the peptide precipitated with diethyl ether. The crude peptides were dissolved in water (50-100 mg/ml) and purified by reverse-phase high-performance liquid chromatography (RP-HPLC). HPLC conditions were: column: Vydac TP21810C18 (10×250 mm); elution system: gradient system of 0.1% TFA in water v/v (A) and 0.1% TFA in acetonitrile (ACN) v/v (B); flow rate 6 ml/minute; absorbance was detected from 190-370 nm. There were different gradient systems used. For example for peptides LQG and LQGV: ten minutes 100% A followed by linear gradient 0-10% B in 50 minutes. For example for peptides VLPALP and VLPALPQ: five minutes 5% B followed by linear gradient 1% B/minute. The collected fractions were concentrated to about 5 ml by rotation film evaporation under reduced pressure at 40° C. The remaining TFA was exchanged against acetate by eluting two times over a column with anion exchange resin (Merck II) in acetate form. The elute was concentrated and lyophilized in 28 hours. Peptides later were prepared for use by dissolving them in PBS.

RAW 264.7 macrophages, obtained from American Type Culture Collection (Manassas, Va.), were cultured at 37° C. in 5% CO₂ using DMEM containing 10% FBS and antibiotics (100 U/ml of penicillin, and 100 μg/ml streptomycin). Cells (1×10⁶/ml) were incubated with peptide (10 μg/ml) in a volume of 2 ml. After eight hours of cultures, cells were washed and prepared for nuclear extracts.

Nuclear extracts and EMSA were prepared according to Schreiber et al., Methods (Schreiber et al. 1989, Nucleic Acids Research 17). Briefly, nuclear extracts from peptide stimulated or nonstimulated macrophages were prepared by cell lysis followed by nuclear lysis. Cells were then suspended in 400 μl of buffer (10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM KCL, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF and protease inhibitors), vigorously vortexed for 15 seconds, left standing at 4° C. for 15 minutes, and centrifuged at 15,000 rpm for two minutes. The pelleted nuclei were resuspended in buffer (20 mM HEPES (pH 7.9), 10% glycerol, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.5 mM PMSF and protease inhibitors) for 30 minutes on ice, then the lysates were centrifuged at 15,000 rpm for two minutes. The supernatants containing the solubilized nuclear proteins were stored at −70° C. until used for the Electrophoretic Mobility Shift Assays (EMSA).

Electrophoretic mobility shift assays were performed by incubating nuclear extracts prepared from control (RAW 264.7) and peptide treated RAW 264.7 cells with a 32P-labeled double-stranded probe (5′ AGCTCAGAGGGGGACTTTCCGAGAG 3′) synthesized to represent the NF-kappaB binding sequence. Shortly, the probe was end-labeled with T4 polynucleotide kinase according to manufacturer's instructions (Promega, Madison, Wis.). The annealed probe was incubated with nuclear extract as follows: in EMSA, binding reaction mixtures (20 μl) contained 0.25 μg of poly(dI-dC) (Amersham Pharmacia Biotech) and 20,000 rpm of 32P-labeled DNA probe in binding buffer consisting of 5 mM EDTA, 20% Ficoll, 5 mM DTT, 300 mM KCl and 50 mM HEPES. The binding reaction was started by the addition of cell extracts (10 μg) and was continued for 30 minutes at room temperature. The DNA-protein complex was resolved from free oligonucleotide by electrophoresis in a 6% polyacrylamide gel. The gels were dried and exposed to x-ray films.

The transcription factor NF-kB participates in the transcriptional regulation of a variety of genes. Nuclear protein extracts were prepared from LPS and peptide treated RAW264.7 cells or from LPS treated RAW264.7 cells. In order to determine whether the peptide modulates the translocation of NF-kB into the nucleus, on these extracts EMSA was performed. Here we see that indeed some peptides are able to modulate the translocation of NF-kB since the amount of labeled oligonucleotide for NF-kB is reduced. In this experiment peptides that show the modulation of translocation of NF-kB are: VLPALPQVVC (SEQ ID NO:_), LQGVLPALPQ (SEQ ID NO:_), LQG, LQGV (SEQ ID NO:1), GVLPALPQ (SEQ ID NO:16), VLPALP (SEQ ID NO:4), VLPALPQ (SEQ ID NO:13), GVLPALP (SEQ ID NO:16), VVC, MTRV (SEQ ID NO:20), MTR.

RAW 264.7 mouse macrophages were cultured in DMEM, containing 10% or 2% FCS, penicillin, streptomycin and glutamine, at 37° C., 5% CO₂. Cells were seeded in a 12-well plate (3×10⁶ cells/ml) in a total volume of 1 ml for two hours and then stimulated with LPS (E. coli 026:B6; Difco Laboratories, Detroit, Mich.,) and/or gene-regulatory peptide (1 microgr/ml). After 30 minutes of incubation, plates were centrifuged and cells were collected for nuclear extracts. Nuclear extracts and EMSA were prepared according to Schreiber et al. Cells were collected in a tube and centrifuged for five minutes at 2000 rpm (rounds per minute) at 4° C. (Universal 30 RF, Hettich Zentrifuges). The pellet was washed with ice-cold Tris buffered saline (TBS pH 7.4) and resuspended in 400 μl of a hypotonic buffer A (10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF and protease inhibitor cocktail (Complete™ Mini, Roche) and left on ice for 15 minutes. Twenty-five microliters 10% NP-40 was added and the sample was centrifuged (two minutes, 4000 rpm, 4° C.). The supernatant (cytoplasmic fraction) was collected and stored at −70° C. The pellet, which contains the nuclei, was washed with 50 μl buffer A and resuspended in 50 μl buffer C (20 mM HEPES pH 7.9, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.5 mM PMSF and protease inhibitor cocktail and 10% glycerol). The samples were left to shake at 4° C. for at least 60 minutes. Finally the samples were centrifuged and the supernatant (nucleic fraction) was stored at −70° C.

Bradford reagent (Sigma) was used to determine the final protein concentration in the extracts. For electrophoretic mobility shift assays (EMSA) an oligonucleotide representing NF-κB binding sequence (5′-AGC TCA GAG GGG GAC TTT CCG AGA G-3′ (SEQ ID NO:_) was synthesized. Hundred pico mol sense and antisense oligo were annealed and labeled with γ-³²P-dATP using T4 polynucleotide kinase according to manufacturer's instructions (Promega, Madison, Wis.). Nuclear extract (5-7.5 μg) was incubated for 30 minutes with 75,000 cpm probe in binding reaction mixture (20 microliters) containing 0.5 μg poly dI-dC (Amersham Pharmacia Biotech) and binding buffer BSB (25 mM MgCl₂, 5 mM CaCl₂, 5 mM DTT and 20% Ficoll) at room temperature. The DNA-protein complex was resolved from free oligonucleotide by electrophoresis in a 4-6% polyacrylamide gel (150 V, two to four hours). The gel was then dried and exposed to x-ray film. The transcription factor NF-kB participates in the transcriptional regulation of a variety of genes. Nuclear protein extracts were prepared from either LPS (1 mg/ml), peptide (1 mg/ml) or LPS in combination with peptide treated and untreated RAW264.7 cells. In order to determine whether the peptides modulate the translocation of NF-kB into the nucleus, on these extracts EMSA was performed. Peptides are able to modulate the basal as well as LPS induced levels of NF-kB. In this experiment peptides that show the inhibition of LPS induced translocation of NF-kB are: VLPALPQVVC (SEQ ID NO:_), LQGVLPALPQ (SEQ ID NO:_, LQG, LQGV (SEQ ID NO:1), GVLPALPQ (SEQ ID NO:16), VLPALP (SEQ ID NO:4), VVC, MTR and circular LQGVLPALPQVVC (SEQ ID NO:17). Peptides that in this experiment promote LPS induced translocation of NF-kB are: VLPALPQ (SEQ ID NO:13), GVLPALP and MTRV. Basal levels of NF-kB in the nucleus was decreased by VLPALPQVVC (SEQ ID NO:_), LQGVLPALPQ (SEQ ID NO:_, LQG and LQGV (SEQ ID NO:1) while basal levels of NF-kB in the nucleus was increased by GVLPALPQ (SEQ ID NO:16), VLPALPQ (SEQ ID NO:13), GVLPALP (SEQ ID NO:16), VVC, MTRV (SEQ ID NO:20), MTR and LQGVLPALPQVVC (SEQ ID NO:17). In other experiments, QVVC also modulated the translocation of NF-kB into the nucleus (data not shown). Further modes of identification of gene-regulatory peptides by NFkB analysis

Cells: Cells will be cultured in appropriate culture medium at 37° C., 5% CO₂. Cells will be seeded in a 12-well plate (usually 1×10⁶ cells/ml) in a total volume of 1 ml for two hours and then stimulated with regulatory peptide in the presence or absence of additional stimuli such as LPS. After 30 minutes of incubation plates will be centrifuged and cells are collected for cytosolic or nuclear extracts.

Nuclear Extracts: Nuclear extracts and EMSA could be prepared according to Schreiber et al. Method (Schreiber et al. 1989, Nucleic Acids Research 17). Cells are collected in a tube and centrifuged for five minutes at 2000 rpm (rounds per minute) at 4° C. (Universal 30 RF, Hettich Zentrifuges). The pellet is washed with ice-cold Tris buffered saline (TBS pH 7.4) and resuspended in 400 μl of a hypotonic buffer A (10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF and protease inhibitor cocktail (Complete™ Mini, Roche) and left on ice for 15 minutes. Twenty-five microliters 10% NP-40 is added and the sample is centrifuged (two minutes, 4000 rpm, 4° C.). The supernatant (cytoplasmic fraction) was collected and stored at −70° C. for analysis. The pellet, which contains the nuclei, is washed with 50 μl buffer A and resuspended in 50 μl buffer C (20 mM HEPES pH 7.9, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.5 mM PMSF and protease inhibitor cocktail and 10% glycerol). The samples are left to shake at 4° C. for at least 60 minutes. Finally the samples are centrifuged and the supernatant (nucleic fraction) is stored at −70° C. for analysis.

Bradford reagent (Sigma) could be used to determine the final protein concentration in the extracts.

EMSA: For Electrophoretic mobility shift assays an oligonucleotide representing NF-κB binding sequence such as (b 5′-AGC TCA GAG GGG GAC TTT CCG AGA G-3′ (SEQ ID NO:_)) are synthesized. Hundred pico mol sense and antisense oligo are annealed and labeled with γ-³²P-dATP using T4 polynucleotide kinase according to manufacturer's instructions (Promega, Madison, Wis.). Cytosolic extract or nuclear extract (5-7.5 μg) from cells treated with regulatory peptide or from untreated cells is incubated for 30 minutes with 75,000 cpm probe in binding reaction mixture (20 μl) containing 0.5 μg poly dI-dC (Amersham Pharmacia Biotech) and binding buffer BSB (25 mM MgCl₂, 5 mM CaCl₂, 5 mM DTT and 20% Ficoll) at room temperature. Or cytosolic and nuclear extract from untreated cells or from cells treated with stimuli could also be incubated with probe in binding reaction mixture and binding buffer. The DNA-protein complex are resolved from free oligonucleotide by electrophoresis in a 4-6% polyacrylamide gel (150 V, two to four hours). The gel is then dried and exposed to x-ray film. Peptides can be biotinylated and incubated with cells. Cells are then washed with phosphate-buffered saline, harvested in the absence or presence of certain stimulus (LPS, PHA, TPA, anti-CD3, VEGF, TSST-1, VIP or know drugs etc.). After culturing cells are lysed and cells lysates (whole lysate, cytosolic fraction or nuclear fraction) containing 200 micro gram of protein are incubated with 50 microliters Neutr-Avidin-plus beads for one hour at 4° C. with constant shaking. Beads are washed five times with lysis buffer by centrifugation at 6000 rpm for one minute. Proteins are eluted by incubating the beads in 0.05 N NaOH for one minute at room temperature to hydrolyze the protein-peptide linkage and analyzed by SDS-polyacrylamide gel electrophoresis followed by immunoprecipitated with agarose-conjugated anti-NF-kB subunits antibody or immunoprecipitated with antibody against to be studied target. After hydrolyzing the protein-peptide linkage, the sample could be analyzed on HPLS and mass-spectrometry. Purified NF-kB subunits or cell lysate interaction with biotinylated regulatory peptide can be analyzed on biosensor technology. Peptides can be labeled with FITC and incubated with cells in the absence or presence of different stimulus. After culturing, cells can be analyzed with fluorescent microscopy, confocal microscopy, flow cytometry (cell membrane staining and/or intracellular staining) or cells lysates are made and analyzed on HPLC and mass-spectrometry. NF-kB transfected (reporter gene assay) cells and gene array technology can be used to determine the regulatory effects of peptides.

HPLC and mass-spectrometry analysis: Purified NF-kB subunit or cytosolic/nuclear extract is incubated in the absence or presence of (regulatory) peptide is diluted (2:1) with 8 N guanidinium chloride and 0.1% trifluoroacetic acid, injected into a reverse-phase HPLC column (Vydac C18) equilibrated with solvent A (0.1% trifluoroacetic acid), and eluted with a gradient of 0 to 100% eluant B (90% acetonitrile in solvent A). Factions containing NF-kB subunit are pooled and concentrated. Fractions are then dissolved in appropriate volume and could be analyzed on mass-spectrometry.

Further Examples

In this study we demonstrate that LQGV (SEQ ID NO:1), AQGV and LAGV (SEQ ID NO:10), administrated after the induction of hemorrhagic shock in rats, significantly reduced TNF-α and IL-6 plasma levels, which is associated with reduced TNF-α and IL-6 mRNA transcript levels in the liver. This indicates that LQGV (SEQ ID NO:1), AQGV (SEQ ID NO:2), LAGV (SEQ ID NO:_may have therapeutic potential with beneficial effects on systemic inflammation, thereby reducing organ integrity/function, which is associated with shock and SIRS often seen with severe trauma patients.

Materials and Methods

Adult Male specific pathogen-free Wistar rats (Harlan C P B, Zeist, N L), weighing 350-400 g were used after a minimum seven-day acclimation period. The animals were housed under barrier conditions and kept at 25° C. with a twelve-hour light/dark cycle. Rats were allowed free access to water and chow (−). All procedures were performed in accordance with the Principles of Laboratory Animal Care (NIH publication No. 86-23, revised 1985) under a protocol approved by the Committee on Animal Research of the Erasmus University (protocol EUR 365).

The rats were fasted overnight but were allowed free access to water before the experiment. Subsequent to endotracheal intubation the rats were mechanically ventilated with an isofluorane (−) N₂O/O₂ mixture at 60 breaths/minute. Body temperature was continuously maintained at 37.5° C. by placing the animals on a thermo controlled “half-pipe” (UNO, NL). Polyethylene tubes (PE-50, Becton Dickinson; St. Michielsgestel, NL) were flushed with heparin and placed via the right carotid artery in the aorta and in the right internal jugular vein. The animals received no heparin before or during the experiment.

Mean arterial pressures (MAP) was measured using transducers (Becton Dickinson) that were connected in line to an electronic recorder (Hewlett Packard, 78354-A Germany) for electronically calculated mean pressures and continuous measurement of the animal's blood pressure. Under semi sterile conditions a median laparotomy was performed and ultrasonic perivascular flow probes (Transonic Systems Inc, Maastricht, NL) were placed on the common hepatic artery and the portal vein. A supra pubic catheter was placed to monitor the urine production during and after resuscitation.

After an acclimatization period of 20 minutes, the rats were randomized into the following five groups:

Hemorrhagic shock group were bled within ten minutes to a mean arterial pressure (MAP) of 40 mmHg and maintained at this level for 60 minutes by withdrawing or re-infusing shed blood as needed. Thereafter, the animals were resuscitated with plus minus four times the volume of the withdrawn blood over 30 minutes with a 0.9% NaCl solution.

The hemorrhagic shock group+peptide A (LAGV; one-letter amino acid code) underwent the same procedure as the hemorrhagic shock group but received a single bolus injection of 5 mg/kg peptide A intravenously 30 minutes after the induction of shock.

The hemorrhagic shock group+peptide B (AQGV) underwent the same procedure as the hemorrhagic shock group and received a single bolus injection of 5 mg/kg peptide B intravenously, 30 minutes after the induction of shock.

The hemorrhagic shock group+peptide C (LQGV) underwent the same procedure as the hemorrhagic shock group and received a single bolus injection of 5 mg/kg peptide C intravenously, 30 minutes after the induction of shock.

Sham group underwent the same procedure as the hemorrhagic shock group without performing the hemorrhage or administration of any kind of peptides.

The hepatic arterial blood flow (QHA) and hepatic portal venous blood flow (QVP) were measured with transit time ultrasonic perivascular flow probes, connected to an ultrasonic meter (T201; Transonic Systems, Inc., Maastricht, NL). Systemic and hepatic hemodynamics were continuously measured. At regular time points arterial blood samples were taken. The animals were euthanized by withdrawal of arterial blood via the carotid artery.

Blood, Tissue, and Cell Harvesting Procedure

Plasma collection and storage: Whole arterial blood was obtained at −15, 30, 60, 90, 120, 150 and 180 minutes after induction of shock via the right carotid artery and collected in duplo. 0.2 ml was placed in tubes (Eppendorf EDTA KE/1.3) to be assayed in the coulter counter (−). 0.5 ml was placed in Minicollect tubes (Bio-one, Greiner) centrifuged for five minutes, immediately frozen, and stored at −80° C., until assayed. All assays were corrected for the hematocrit.

Measurement of cytokines (still in progress): The levels of IL-6, and IL-10 in the serum were determined by an ELISA (R&D Systems Europe Ltd) according to the manufacturer's instructions.

Histology (still in progress): The alterations in lung, liver, sigmoid and small bowel morphology were examined in sham-operated animals, in animals after trauma-hemorrhage and in animals after trauma-hemorrhage treated with peptide A, B or C. All tissues were collected in duplo. One part was harvested and fixed in formalin (Sigma) and later embedded in paraffin. The other part was placed in tubes (NUNC Cryo tube™ Vials), quick frozen in liquid nitrogen and stored at −80° C. until assayed.

Results

Mean Arterial Pressure: MAP dropped in all shock groups significant during the shock phase compared to the control group.

Hematocrit: The hematocrit following trauma-hemorrhage was similar in the different peptide A, B and C treated and non-treated groups. During the shock phase there was a difference of hematocrit in the control group in comparison with the other groups. From the resuscitation phase (90 minutes) there was no significant difference in hematocrit among the control, trauma-hemorrhage, and peptide groups.

Leukocyte Recruitment: During trauma-hemorrhage the leukocytes dropped from 100% at T0 in all groups to a minimum of 40.0±11.9%, 42.0±8.7%, 47.3±12.4%, 38.2±7.4% in respectively the non-treated, peptide A treated, peptide B treated and peptide C treated group because of leukocyte accumulation in the splanchnic microcirculation. There was a significant difference in leukocyte concentration between all treated and non-treated trauma-hemorrhage groups, and the control group during the shock phase. No significant difference was noticed between the peptide A, B or C treated animals and the non-treated animals.

Blood Concentrations Of Macrophages And Granulocytes: At 180 minutes after the onset of trauma-hemorrhage, concentrations of circulating macrophages (M_(Φ)) and granulocytes were significant lower in the peptide B and C treated animals compared with the corresponding experimental group. Blood levels of circulating M_(Φ) and granulocytes were 5,556±1,698 10⁹/l in sham-operated animals whereas blood levels were 6,329±1,965 10⁹/l after trauma-hemorrhage, and decreased by 29.9% after administration of peptide B (4,432±0.736 10⁹/l) and 39.2% after administration of peptide C (3,846±0.636 10⁹/l) compared with concentrations after trauma-hemorrhage.

Arterial Hepatic Blood Flow: There was a decrease in the arterial hepatic blood flow in the shock group (18.3±14.3%) and in the peptide A (21.3±9.1%), B (18.1±9.0%) and C (21.2±8.6%) group during the shock period compared with the control group (102.6±23.5%). An increase in blood flow was observed during the reperfusion in the hepatic artery of the shock group (128.9±75.4%) compared with control animals (83.7±24.2%) and the animals treated with peptide B (78.4±28.3%).

Trauma-hemorrhage results in hypoxic stress owing to the absolute reduction in circulating blood volume. In contrast, sepsis is an inflammatory state mainly mediated by bacterial products. It is interesting that these divergent insults reveal similar pathophysiologic alterations in terms of the splanchnic circulation.

Hemorrhagic shock significantly increases leukocyte accumulation in the splanchnic microcirculation owing to the up-regulation of P selectin. The expression of intercellular adhesion molecule within the intestinal muscular vasculature after hemorrhagic shock promotes the local recruitment of leukocytes, and this inflammatory response is accompanied by subsequent impairment of intestinal function.

The adhesion and extravasation of neutrophils not only contribute to the inflammatory response in the splanchnic tissue bed but also induce intestinal microcirculatory failure and dysfunction after severe stress. This is mediated by the induced expression of adhesion molecules, such as selectins and endothelial cell adhesion molecules, on the surface of neutrophils and endothelial cells.

In our shock experiments, leukocyte concentration significant decreases during hemorrhagic shock compared to the control animals. However a single dose of peptide B or C administered during resuscitation, decreased concentrations of circulating macrophages and granulocytes 120 minutes after the onset of hemorrhagic shock compared to the non-treated animals.

Because some female sex hormones effectively protect the organs from circulatory failure after various adverse circulatory conditions, numerous studies have been performed to clarify the molecular mechanism of for example estradiol action with regard to tissue circulation. In this study, a single dose of peptide was administered following trauma-hemorrhage and various parameters were measured at three hours following the induction of sepsis. Treatment with peptides improved or restored immune functional parameters and cardiovascular functions. Therefore, our results show that administration of short oligopeptides (NMPFs) is beneficial in the treatment of critically ill trauma victims experiencing hemorrhagic shock.

Example 2

BACKGROUND: Hemorrhagic shock followed by resuscitation induces a massive pro-inflammatory response, which may culminate into severe inflammatory response syndrome, multiple organ failure and finally death. Treatments aimed at inhibiting the effects of pro-inflammatory cytokines are only effective when initiated before the onset of hemorrhagic shock, which severely limits their clinical application.

AIM: We investigated whether the administration of synthetic hCG-related oligopeptides (LQGV (SEQ ID NO:1), AQGV (SEQ ID NO:2), LAGV (SEQ ID NO:10)) 30 minutes after induction of hemorrhagic shock reduced the inflammatory response.

METHODS: Rats were bled to 50% of baseline mean arterial pressure and one hour later resuscitated by autologous blood transfusion. Thirty minutes after onset of hemorrhagic shock, experimental groups received either one of the synthetic hCG-related oligopeptides (LQGV (SEQ ID NO:1), AQGV (SEQ ID NO:2), LAGV (SEQ ID NO:10)) or 0.9% NaCl solution. TNF-cc and IL-6 plasma levels were determined at fixed time points before and after onset of hemorrhagic shock. Liver, lungs, ileum and sigmoid mRNA levels for TNF-α, IL-6 and ICAM-1 were determined 180 minutes after onset of hemorrhage.

RESULTS: Treatment with either one of the three hCG-related oligopeptides (LQGV (SEQ ID NO:1), AQGV (SEQ ID NO:2), LAGV (SEQ ID NO:10)) efficiently reduced TNF-α and IL-6 plasma levels as well as TNF-α and IL-6 mRNA transcript levels in the liver.

CONCLUSION: Considering these powerful effects of hCG-related oligopeptides during severe hemorrhagic shock, they may have therapeutic potential with beneficial effects on the hyper inflammation, thereby reducing the late life threatening tissue- and organ-damage that is associated with severe hemorrhagic shock.

INTRODUCTION: In hemorrhagic shock there is massive blood loss, which cannot be compensated by the body without treatment. The primary treatment of hemorrhagic shock is to control bleeding and restore intravascular volume to improve tissue perfusion. This treatment induces an inflammatory response, which may culminate into a severe inflammatory response and finally multiple organ dysfunction syndrome (MODS).^([1, 2, 3]) In addition, approximately 40% of patients develop sepsis as a result of trauma-hemorrhage.[³] Sepsis and MODS are the leading causes of death in critically ill patients on the intensive care unit all over the world with mortality rates of about 50%.^([4, 5])

The severe inflammatory response due to trauma-hemorrhage is characterized by increased expression of adhesion molecules, such as intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), on sinusoidal endothelial cells and hepatocytes. Furthermore, increased levels of pro-inflammatory cytokines are found systemically and locally in liver, lungs and intestine.^([6, 7, 8, 9]) The pro-inflammatory cytokines produced are in particular tumor necrosis factor alpha (TNF-α), interleukin (IL)-1β and IL-6.^([10, 11, 12]) These cytokines affect organ integrity/function directly, but also indirectly through secondary mediators, such as nitric oxide, thromboxanes, leukotrienes, platelet-activating factor, prostaglandins, and complement.^([13, 14]) TNF-α also causes the release of tissue-factor by endothelial cells leading to fibrin deposition and disseminated intravascular coagulation.^([15, 16]) Cells within the liver, mainly Kupffer cells, but also hepatocytes and sinusoidal endothelial cells, are considered as the main producers of these pro-inflammatory cytokines during hemorrhagic shock.^([17])

The last decade, researchers have focused on the modulation of the systemic inflammatory responses with therapeutic agents aiming at neutralizing the activity of cytokines, especially TNF-α.^([18]) Other researchers used therapeutic agents aiming at the inhibition of TNF-α production.^([19]) However, most of these therapeutic agents must be administered before the onset of hemorrhagic shock to achieve a therapeutic effect.^([19]) Clearly, this is almost impossible in a clinical trauma-hemorrhage setting. Therefore, therapies initiated after the onset of severe trauma-hemorrhage and aiming at reducing the production of pro-inflammatory cytokine are more relevant to prevent the events leading to MODS.

During pregnancy, the maternal immune system tolerates the fetus by reducing the cell-mediated immune response while retaining normal humoral immunity.^([20]) Also, clinical symptoms of cell-mediated autoimmune diseases regress in many patients during pregnancy.^([20]) The hormone human chorionic gonadotropin (hCG) is mainly secreted by placental syncytiocytotrophoblasts during pregnancy and has been shown to be immunoregulatory^([21, 22, 23]) The β-subunit of hCG is degraded by specific proteolytic enzymes.^([24]) This can lead to the release of several oligopeptides consisting of four to seven amino acids which, because of their role in regulation of physiological processes, are considered regulatory.^([25]) We successfully demonstrated that synthetic hCG-related oligopeptides can inhibit the acute inflammatory response, disease severity, and mortality in high-dose lipopolysaccharide induced systemic inflammatory response syndrome.^([26]) Considering these powerful regulating effects of synthetic hCG-related oligopeptides on inflammation, we hypothesized that the administration of such regulatory oligopeptides after severe trauma-hemorrhage could inhibit the massive inflammatory response, associated with this condition. To this end, we used LQGV (SEQ ID NO:1), which is part of the primary structure of loop two of the β-subunit of hCG, and two alanine replacement variants, namely AQGV (SEQ ID NO:2) and LAGV (SEQ ID NO:10).

In this study we demonstrate that LQGV (SEQ ID NO:1), AQGV (SEQ ID NO:2), and LAGV (SEQ ID NO:10), administrated after the induction of hemorrhagic shock in rats, significantly reduced TNF-α and IL-6 plasma levels, which is associated with reduced TNF-α and IL-6 mRNA transcript levels in the liver. This indicates that LQGV (SEQ ID NO:1), AQGV (SEQ ID NO:2), LAGV (SEQ ID NO:10) may have therapeutic potential with beneficial effects on systemic inflammation, thereby reducing organ integrity/function, which is associated with severe hemorrhagic shock.

Materials and Methods Animals

Adult male specific pathogen-free Wistar rats (Harlan CPB, Zeist, NL), weighing 350-400 g were used. Animals were housed under barrier conditions at 25° C. with a twelve-hour light/dark cycle, and were allowed food and water ad libitum. The experimental protocol was approved by the Animal Experiment Committee under the Dutch Experiments on Animals Act and adhered to the rules laid down in this national law that serves the implementation of “Guidelines on the protection of experimental animals” by the Council of Europe (1986), Directive 86/609/EC.

hCG-related synthetic oligopeptides: The hCG-related oligopeptides (LQGV (SEQ ID NO:1), AQGV (SEQ ID NO:2), and LAGV (SEQ ID NO:10)) were synthesized by Ansynth Service B.V. (Roosendaal, NL) and dissolved in 0.9% NaCl at a concentration of 10 mg/ml.

Surgical procedures: Rats were food deprived overnight before the experiment, but were allowed water ad libitum. Rats were anesthetized using a mixture of N₂O/O₂ isoflurane (Pharmachemie B.V., Haarlem, NL). Body temperature was continuously maintained at 37.5° C. by placing the rats on a thermo controlled “half-pipe” (UNO, Rotterdam, NL). Endotracheal intubation was performed, and rats were ventilated at 60 breaths per minute with a mixture of N₂O/O₂ 2% isoflurane. Polyethylene tubes (PE-50, Becton Dickinson; St. Michielsgestel, NL) were flushed with heparin and placed via the right carotid artery in the aorta and in the right internal jugular vein. The rats received no heparin before or during the experiment.

Experimental procedures: After an acclimatization period of 15 minutes, the rats were randomized into five different groups: 1) sham, 2) hemorrhagic shock (HS), 3) hemorrhagic shock with LQGV (SEQ ID NO:1) treatment (HS/LQGV (SEQ ID NO:1)), 4) hemorrhagic shock with AQGV (SEQ ID NO:2) treatment (HS/AQGV (SEQ ID NO:2)) and 5) hemorrhagic shock with LAGV (SEQ ID NO:10) treatment (HS/LAGV (SEQ ID NO:10)). Hemorrhagic shock was induced by blood withdrawal, reducing the circulating blood volume until a mean arterial pressure (MAP) of 50% of normal mmHg was reached. This level of hypotension was maintained for 60 minutes. After 30 minutes, rats received either a single bolus injection of 10 mg/kg LQGV (SEQ ID NO:1), AQGV (SEQ ID NO:2), LAGV (SEQ ID NO:10) or 0.9% NaCl solution. The peptides and dosage were based on previous studies, in which we performed dose-escalation experiments (manuscript in preparation). Sixty minutes after induction of hemorrhagic shock, rats were resuscitated by autologous blood transfusion over a period of 30 minutes and monitored for another 120 minutes after which they were sacrificed (FIG. 7A). Sham animals underwent the same surgical procedure as the hemorrhagic shock animals, but without performing hemorrhage and administration of peptides.

Plasma collection and storage: Arterial blood was obtained 15 minutes before and 30, 60, 90, 120, 150 and 180 minutes after onset of hemorrhage (FIG. 7A). After blood withdrawal, leukocyte numbers were determined using a coulter counter (Beckman Coulter, Mijdrecht, NL) and corrected for the hematocryte. Approximately, 0.3 ml of blood was placed into mini collect tubes (Greiner, Bio-one, Alphen a/d Rijn, NL), plasma was obtained by centrifugation (1,500 rpm; five minutes), immediately frozen, and stored at −80° C., until assayed.

Measurements of Mean arterial pressure: During the experiments, mean arterial pressure (MAP) was continuously measured using transducers (Becton Dickinson) that were connected in line to an electronic recorder (Hewlett Packard, 78354-A, Germany).

Tissue collection and storage: Liver, lungs, ileum and sigmoid were surgically removed at the end of the experiment, snap-frozen, and stored at −80° C., until assayed.

Measurement of cytokines: TNF-α and IL-6 plasma levels were determined by ELISA (R&D Systems Europe Ltd, Abingdon, UK), according to the manufacturer's instructions.

Evaluation of mRNA levels by real-time quantitative (RQ)-PCR: RNA was isolated using a QIAGEN kit (QIAGEN, Hilden, Germany), according to the manufacturer's instructions. TNF-α, IL-6 and ICAM-1 transcripts were determined by RQ-PCR using an Applied Biosystems 7700 PCR machine (Foster City, Calif., USA) as described previously.^([27]) TNF-α, IL-6 and ICAM-1 expression was quantified by normalization against GAPDH. Primer probe combinations used are listed in Table 1.

Statistical analysis: Statistical analysis was performed using SPSS version 11 software (SPSS Inc., Chicago, Ill). Inter group differences were analyzed with Kruskal-Wallis statistical test. If Kruskal-Wallis statistical testing resulted in a p<0.05, a Dunn's Multiple Comparison test was performed and p <0.05 was considered statistically significant.

Results

Induction of hemorrhagic shock: Lowering the MAP to 50% of normal induced hemorrhagic shock, which was successfully maintained for 60 minutes in all four experimental groups (FIG. 7B). No change in MAP was observed in sham treated rats (FIG. 7B). A decrease in the percentage of blood leukocytes was observed in all four experimental groups after blood withdrawal (FIG. 7C). Sixty minutes after hemorrhagic shock, rats were resuscitated with there own blood to induce organ reperfusion, which was associated with a normalization of leukocyte level (FIG. 7C).

Oligopeptide treatment reduces pro-inflammatory cytokine plasma levels: The therapeutic capacity of three synthetic oligopeptides (LQGV, AQGV (SEQ ID NO:2), LAGV) related to the primary structure of loop two of the β-subunit of hCG was evaluated in a rat hemorrhagic shock model. Before induction of hemorrhage, TNF-α plasma levels were comparable in all five groups (˜15-24 μg/ml) (FIG. 8). In the HS group, TNF-α levels started to increase thirty minutes after induction of hemorrhagic shock and were significantly increased after sixty minutes, as compared to the sham group (264 pg/ml vs 24 pg/ml, respectively; p<0.01). TNF-α levels reached a maximum of 374 pg/ml after 90 minutes in the HS group, after which levels declined again but always remaining increased compared to the sham group (FIG. 8). In contrast, none of the oligopeptide-treated HS groups (HS/LQGV, HS/AQGV, HS/LAGV) showed an increase in plasma TNF-α levels during the experiment (FIG. 8). IL-6 levels are known to increase at a later time-point than TNF-α after severe hemorrhagic shock.^([11, 12]) Therefore, we determined IL-6 levels in blood samples collected 120, 150 and 180 minutes after the onset of hemorrhagic shock. In the HS group, IL-6 plasma levels were significantly increased as compared to sham group at 120 minutes (1704 pg/ml vs 338 pg/ml, respectively; p<0.001), at 150 minutes (2406 pg/ml vs 316 pg/ml, respectively; p<0.001) and at 180 minutes (2932 pg/ml vs 369 pg/ml, respectively; p<0.001) (FIG. 9). Although IL-6 levels tended to increase a little in the HS/oligopeptide treated rats as compared to sham treated rats, this never reached significance. Treatment with oligopeptides after hemorrhagic shock (HS/LQGV (SEQ ID NO:1), HS/AQGV (SEQ ID NO:2), HS/LAGV (SEQ ID NO:10)) resulted in a significant reduction of IL-6 plasma levels as compared to the non-treated hemorrhagic shock group (HS) (FIG. 9). These data demonstrate that treatment with a single dose of either LQGV (SEQ ID NO:1), AQGV (SEQ ID NO:2), or LAGV (SEQ ID NO:10) after induction of hemorrhagic shock results in a significant reduction of TNF-α and IL-6 plasma levels.

Oligopeptide treatment reduces TNF-α and IL-6 but not ICAM-1 mRNA levels in the liver: Because oligopeptide treatment clearly decreased the TNF-α and IL-6 plasma levels, we analyzed mRNA levels in liver, lungs, ileum and sigmoid tissues at 180 minutes after the onset of hemorrhagic shock. In the liver, TNF-α transcripts were significantly increased in the HS group as compared to the sham group. Oligopeptide treatment was associated with decreased TNF-α transcripts in the liver as compared to non-treated HS rats with only HS/LQGV showing a significant reduction as compared to HS (p<0.01; FIG. 10A).

In the HS group, IL-6 transcripts in the liver were increased ˜83 times as compared to the sham group (p<0.001; FIG. 10B). None of the oligopeptide treated groups showed an increase in IL-6 mRNA as compared to the sham treated group. LQGV (SEQ ID NO:1) and AQGV (SEQ ID NO:2) treatment resulted in a significant reduction in IL-6 mRNA transcripts as compared to the HS group (p<0.05; FIG. 10B).

ICAM-1 transcript levels in the liver were significantly increased in the HS group as compared to the sham group (FIG. 10C). Oligopeptide treatment during hemorrhagic shock (HS/LQGV (SEQ ID NO:1), HS/AQGV (SEQ ID NO:2), HS/LAGV (SEQ ID NO:10)) did not affect the ICAM-1 transcript levels in the liver (FIG. 10C). In lungs, ileum and sigmoid tissue no significant differences could be detected between the various groups for TNF-α, IL-6 and ICAM-1 (data not shown). These data indicate that oligopeptide treatment following hemorrhagic shock decreases pro-inflammatory cytokine transcript levels in the liver but does not reduce ICAM-1 transcript levels.

Discussion

In this study we used a rat model of hemorrhagic shock and demonstrated that administration of synthetic hCG-related oligopeptides (LQGV (SEQ ID NO:1), AQGV (SEQ ID NO:2), LAGV (SEQ ID NO:10)) 30 minutes after shock induction, efficiently reduces the pro-inflammatory cytokine levels associated with this condition. Our data demonstrate this to be the consequence of reduced expression of pro-inflammatory cytokine mRNA transcript levels in the liver.

Hemorrhagic shock is associated with an early adherence of leukocytes to the vascular endothelium as a result of a decreased blood volume.^([28]) In our model a decrease in the percentage of leukocytes was detected in all four experimental groups after blood withdrawal. This indicates that all experimental groups experienced hemorrhagic induced shock. Resuscitation resulted in an increase of the percentages of leukocytes in the experimental groups.

Hemorrhagic shock followed by resuscitation induces a severe inflammatory response, which is characterized by an exaggerated production of early pro-inflammatory cytokines, such as TNF-α, IL1β, and subsequently IL-6.^([10, 11, 12]) TNF-α is a key mediator of the innate immune system that is crucial for the generation of a local protective immune response against infectious or non-infectious agents.^([9]) However, uncontrolled massive TNF-α production is lethal, as it spreads via the bloodstream into other organs thereby inducing tissue damage and promoting the production of secondary pro-inflammatory mediators, such as IL6.^([10, 11])

Despite improvement in treatment strategies, trauma-hemorrhage patients may still develop severe inflammatory response that leads too MODS and finally death. Experimental treatment strategies aimed at neutralizing bioactive cytokines, such as monoclonal antibodies against TNF-α, have been successfully applied in several inflammatory disorders, including Crohn's disease and Rheumatoid Arthritis.^([29, 30)] However, clinical studies using monoclonal antibodies against TNF-α showed no clinical effect in trauma-patients.^([31]) It has been suggested that TNF-α neutralizing antibodies causes the accumulation of a large pool of TNF-α/anti-TNF-α pool, which act as a slow release reservoir that may lead to increased constant active TNF-α.^([32]) Therefore, aiming at therapies that decrease the production of TNF-α and IL-6 may be more beneficial in limiting tissue damage and mortality rates in trauma-hemorrhage patients than neutralization of already produced cytokines.

In hemorrhagic shock, TNF-α is secreted within minutes after cellular stimulation, while production stops after three hours, and TNF-α plasma levels become almost undetectable.1 ⁹ 1 We demonstrate that hCG-related regulatory oligopeptides (LQGV (SEQ ID NO:1), AQGV (SEQ ID NO:2), LAGV (SEQ ID NO:10)), administered 30 minutes after the induction of hemorrhagic shock, significantly reduced TNF-α and IL-6 plasma levels. Whether the effect on IL-6 production is direct, or indirect due to reduced TNF-α plasma levels cannot be concluded from our data. Nevertheless, establishing a reduction of IL-6 is of clinical importance, because high IL-6 plasma levels correlate with poor outcome and decreased survival in patients with severe trauma and infection.^([33, 34]) Cells within the liver, are considered as the main producers of pro-inflammatory cytokines during hemorrhagic shock.^([17]) TNF-α and IL-6 transcript levels were significantly increased in the livers of the HS group. LQGV (SEQ ID NO:1), AQGV (SEQ ID NO:2), or LAGV (SEQ ID NO:2) treatment was associated with a reduction in TNF-α and IL-6 liver transcripts, which may be indicative of decreased transcriptional activation. Another important characteristic of endothelial cells and hepatocytes during hemorrhagic shock is increased expression of the adhesion molecule ICAM-1.^([7, 8]) Our study confirms the increased ICAM-1 expression in the liver after hemorrhagic shock. However, LQGV (SEQ ID NO:1), AQGV (SEQ ID NO:2), or LAGV (SEQ ID NO:10) treatment did not result in reduced ICAM-1 expression. This could be due to the inability of hCG-related oligopeptides to interfere with induction of ICAM-1 transcription. In lungs, ileum and sigmoid, we detected no effect of hemorrhagic shock on the induction of TNF-α, IL-6 and ICAM-1 transcripts. This confirms that the liver is the first organ in which the inflammatory response is initiated after hemorrhagic shock and fluid resuscitation.^([15, 31, 32]) In summary, a single administration of a synthetic hCG-related oligopeptide (LQGV (SEQ ID NO:1), AQGV (SEQ ID NO:2), LAGV (SEQ ID NO:10)) after the induction of severe trauma-hemorrhage reduces the subsequent pro-inflammatory response. These data suggest that these oligopeptides have therapeutic potential, in minimizing the late life threatening tissue- and organ-damage that is associated with SIRS seen with severe trauma.

Example Treatment of Severe Skin Inflammations

To assess the activity of the various peptides with skin inflammations an animal model was developed in which these inflammations are generated via topical application of an inflammatory agent to the skin of experimental mice. For this purpose mice were treated with 4% or 5% imiquimod. Imiquimod is an immune response modifier. It is manufactured as a 4% or 5% cream (ALDARA™). Imiquimod works by stimulating the immune system to release a number of chemicals called cytokines whereby it results in inflammation. The imiquimod is taken up by the so-called “toll-like receptor 7” on certain immune cells that are found in the outside part of the skin, the epidermis. Skin areas treated with imiquimod will be come inflamed. The effects include itching, burning, redness, ulceration (sores), scabbing, flaking and pain. Particularly the mice treated with the 5% cream developed intense inflammatory skin lesions.

Peptides tested in this study were Peptide A (LAGV (SEQ ID NO:10)); Peptide B (AQGV (SEQ ID NO:2)); Peptide G (VLPALPQ (SEQ ID NO:13)) and Peptide I (LQGV). Peptides were given parenterally by intraperitoneal injection (i.p.). All peptides had beneficial activity on the imiquimod induced skin lesions, especially after the lesions had occurred (see FIGS. 11 and 12). Treatment with petroleum ether to remove fat and scales of the imiquimod induced lesions in one experiment improved the activity of peptide I (LQGV (SEQ ID NO:1)).

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Further References

WO 99/59671,

WO 01/72831,

WO 97/49721,

WO 01/10907, and

WO 01/11048.

The contents of entirety of each of the references cited herein is incorporated in their entireties by this reference. 

What is claimed is:
 1. A hypertonic pharmaceutical composition for topical application comprising a gene-regulatory peptide or functional analogue thereof.
 2. The hypertonic pharmaceutical composition of claim 1, comprising a peptide selected from the group consisting of LQG, AQG, LQGV (SEQ ID NO:1), AQGV (SEQ ID NO:2), LQGA (SEQ ID NO:3), VLPALP (SEQ ID NO:4), ALPALP (SEQ ID NO:5), VAPALP (SEQ ID NO:6), ALPALPQ (SEQ ID NO:7), VLPAAPQ (SEQ ID NO:8), VLPALAQ (SEQ ID NO:9), LAGV (SEQ ID NO:10), VLAALP (SEQ ID NO:11), VLPALA (SEQ ID NO:12), VLPALPQ (SEQ ID NO:13), VLAALPQ (SEQ ID NO:14), VLPALPA (SEQ ID NO:15), GVLPALP (SEQ ID NO:16), LPGC (SEQ ID NO:_), MTRV (SEQ ID NO:20), MTR, and VVC.
 3. The hypertonic pharmaceutical composition of claim 2, comprising a peptide selected from the group consisting of LAGV (SEQ ID NO:10), AQGV (SEQ ID NO:2), and LQGV (SEQ ID NO:1). 